( c , d ) Primary cortical neurons were transduced with a control lentivirus ( FIGB ) or with one expressing humanNDP52 ( hNDP52 ) at DIV 1 . To induce autophagy , trehalose ( 150 mM ) was added at DIV 5 and the neurons incubated for 24 h ( DIV 6 ) . Primary cortical neurons were fixed with 4 % paraformaldehyde , and stained with the 12E8 or PHF1 antibodies . Scale bar , 20 μm . The optical density of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) . ( e ) and Ser396 / Ser404 ( PHF1 ) ( f ) in the soma of ~ 30 neurons randomly chosen was analysed with the ImageJ program . Data were analysed using Student ' s t - test ( * * * P0 . 001 ) .

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(c,d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing humanNDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar, 20 μm. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons randomly chosen was analysed with the ImageJ program. Data were analysed using Student's t-test (***P0.001).


( b ) Naive cortical cells were co - transfected with the plasmids expressing HA - hNDP52 or HA - tagged domain - deleted mutants of human NDP52 together with GFP - tau . Twenty - four hours later , cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA - hNDP52 constructs and GFP - tau . Homogenates ( input ) used for co - immunoprecipitation were analysed by immunoblotting using anti - HA or a polyclonal tau - specific antibody ( Tau ) .

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(b) Naive cortical cells were co-transfected with the plasmids expressing HA-hNDP52 or HA-tagged domain-deleted mutants of human NDP52 together with GFP-tau. Twenty-four hours later, cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA-hNDP52 constructs and GFP-tau. Homogenates (input) used for co-immunoprecipitation were analysed by immunoblotting using anti-HA or a polyclonal tau-specific antibody (Tau).


( c ) Stably transfected CN1 . 4 cortical cells were maintained in the presence of doxycycline ( 1 μg ml − 1 ) to induce the expression of tau for 24 h . Homogenates from CN1 . 4 cortical cells were used for the co - immunoprecipitation of mouse NDP52 ( mNDP52 ) using a Tau5 antibody . Co - precipitation of mNDP52 was examined by immunoblotting using anti - NDP52 antibody . Input of the homogenates used for co - immunoprecipitation was analysed by immunoblotting using anti - NDP52 or Tau antibody .

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(c) Stably transfected CN1.4 cortical cells were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h. Homogenates from CN1.4 cortical cells were used for the co-immunoprecipitation of mouse NDP52 (mNDP52) using a Tau5 antibody. Co-precipitation of mNDP52 was examined by immunoblotting using anti-NDP52 antibody. Input of the homogenates used for co-immunoprecipitation was analysed by immunoblotting using anti-NDP52 or Tau antibody.


( d , e ) CN1 . 4 cortical cells were transfected with the mock or HA - mNDP52 plasmid ( d ) or with mNDP52 siRNA or scramble RNA as a control ( e ) . The cells transfected with siRNA or scramble RNA were treated with SFN ( 10 μM ) for 24 h . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,e) CN1.4 cortical cells were transfected with the mock or HA-mNDP52 plasmid (d) or with mNDP52 siRNA or scramble RNA as a control (e). The cells transfected with siRNA or scramble RNA were treated with SFN (10 μM) for 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot.


( a ) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co - immunoprecipitation of human NDP52 ( hNDP52 ) using a Tau5 , 12E8 or PHF1 antibody .

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(a) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co-immunoprecipitation of human NDP52 (hNDP52) using a Tau5, 12E8 or PHF1 antibody.


( b ) Homogenates used for co - immunoprecipitation in a were analysed by immunoblotting using anti - NDP52 , Tau5 , 12E8 or PHF1 antibody .

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(b) Homogenates used for co-immunoprecipitation in a were analysed by immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody.


( c ) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl - soluble and insoluble fractions . The levels of total tau and tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 in the sarkosyl - insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies , respectively . Samples were also immunoblotted with a polyclonal to total tau ( Tau ) antibody . The levels of hNDP52 , p62 / SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( d - f ) The regression graphs show the relative amount of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( d ) , at Ser396 / Ser404 ( PHF1 ) ( e ) or total tau ( f ) compared to hNDP52 present in the sarkosyl - insoluble fractions .

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(c) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of total tau and tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting. The relative molecular masses (kDa) are indicated to the left of each blot. (d-f) The regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356 (12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to hNDP52 present in the sarkosyl-insoluble fractions.


( C ) Competition of sybodies and ACE2 for spike protein binding , investigated by GCI . Biotinylated spike protein was immobilized on the GCI sensor and then Sb # 15 ( 200 nM , left ) , or Sb # 68 ( 200 nM , right ) were injected alone or premixed with human ACE2 ( 100 nM ) . Sb # 0 represents a non - randomized control sybody .

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(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.


Neutralization assays using VSVΔG pseudotyped with wildtype SARS - CoV - 2 spike protein . Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs . ( A ) Sb # 15 , Sb # 68 , or an equimolar mixure of both sybodies . ( B ) Sb # 14 , Sb # 16 , Sb # 45 , or , as a positive control , the previously - described sybody MR3 . ( C ) Neutralization by bivalent sybody - Fc fusions . Error bars correspond to standard deviations of three biological replicates .

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Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.


( C ) Neutralization assay using VSVΔG pseudotyped with wildtype Spike - protein . Relative infectivity was determined in response to increasing GS4 or Tripod - GS4r concentration . The corresponding neutralization data for isolated Sb # 15 and Sb # 68 are provided as reference . ( D ) Control experiment using VSVΔG pseudotyped with VSV - G . Infectivity was not affected by Tripod - GS4r .

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(C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r.


( C ) Neutralization assay using VSVΔG pseudotyped with Spike - protein containing the Q493R or P384H mutation , respectively . Relative infectivity in response to increasing binder concentrations was determined .

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(C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined.


( A ) GCI - based kinetic analysis of interactions between immobilized sybodies ( indicated above each plot ) and wild - type ( WT ) or mutant RBDs carrying the individual K417N , E484K or N501Y mutations ( left panels ) , the combined triple KEN ( Beta ) mutations ( middle panels ) , or the double L452R / T478K ( Delta ) mutations ( right panels ) . Sb # 15 , Sb # 68 and GS4 were immobilized on independent flow - cells via biotinylated Avi - tags , and the RBD variants were sequentially injected at a concentration of 200 nM .

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(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.


B Silver stain of the GST ‐ PTB RRM2pull ‐ down of the wild ‐ type RRM2 and the Y247Q mutant . Five microlitre of the pull ‐ down was run on a 15 % SDS - PAGE and silver ‐ stained . Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull ‐ down . The region encompassing 50 kDa to the top of the gel was sliced and subjected to in ‐ gel digestion and mass spectrometry . The two strongest bands visible in this region are labelled as Matrin3 and Raver1 .

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B Silver stain of the GST‐PTB RRM2pull‐down of the wild‐type RRM2 and the Y247Q mutant. Five microlitre of the pull‐down was run on a 15% SDS-PAGE and silver‐stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull‐down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in‐gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1.


C Proteins identified in the GST ‐ PTB RRM2 pull ‐ down ranked by their unique peptide number . The table shows the different proteins we found binding to RRM2 , as well as when present , the sequence of the PRI motif . The indicated function is only a guideline as many have more functions than shown .

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C Proteins identified in the GST‐PTB RRM2 pull‐down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown.


D Western blot of the GSTpull ‐ down using antibodies against Matrin3 and Raver1 . Lanes 1 and 2 show 5 and 10 % of input , respectively , lanes 3 and 4 show GST ‐ PTB full ‐ length pull ‐ down of wild ‐ type and Y247Q mutant , respectively , and lanes 5 and 6 show GST ‐ PTB RRM2 pull ‐ down of wild ‐ type and Y247Q mutant , respectively , and lane 7 with pull ‐ down using GST alone .

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D Western blot of the GSTpull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.


B FLAGimmunoprecipitation of Matrin3 and Raver1 , both with wild ‐ type and with PRI mutated , and FLAG ‐ MS2 as a negative control . The immunoprecipitated complex was separated in a SDS - PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild ‐ type Matrin3 ( lane 2 ) and Raver1 ( lane 4 ) . The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins .

B FLAGimmunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS-PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins.


C The 20 ‐ residue Raver1 491 - 511 ( lane 1 ) and the Matrin3 346 - 365 ( lane 3 ) peptide fused to MS2 were transcribed and translated in vitro ( Input ) and then pulled down with GST ‐ PTB or with GST ‐ SXL as a control . Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested ( lane 4 ) .

C The 20‐residue Raver1 491-511 (lane 1) and the Matrin3 346-365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).


A Western blot probed for Matrin3 ( top panel ) , PTB ( middle panel ) and actin ( lower panel ) . Lanes 1 - 4 contain a twofold dilution of the control C2 sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % , lane 4 - 100 % ) . Lanes 4 - 7 contain equal amount of proteins , as can be confirmed by the anti ‐ actin ( lower panel ) , of control sample ( lane 4 ) , double ‐ PTB and nPTB siRNA ‐ treated sample ( lane 5 ) , Matrin3 siRNA ‐ treated sample ( lane 6 ) and triple knockdown of Matrin3 , PTB and nPTB ( lane 7 ) . Lane 7 is from the same gel and exposure , but some lanes present in the original gel were cropped for clarity , and a black line to indicate cropping was placed .

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A Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1-4 contain a twofold dilution of the control C2 sample (lane 1-12.5%, lane 2-25%, lane 3-50%, lane 4-100%). Lanes 4-7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed.


F RT - PCR validation of Matrin3 ‐ regulated alternative spicing events in the ST7 , ACSL3 , PLEKHA3 , TCF12 , VWA5A , PTBP2 , PTBP3 , C3orf17 , ZMYND8 , VEZT , PIGX and DMD genes . In each case , triplicates for each condition ( C - control , M - Matrin3 , PTB / nPTB and Matrin3 / PTB / nPTB siRNA transfection samples ) were analysed and exon inclusion ( EI ) percentage is shown beneath the corresponding lane , along with the standard deviation ( s . d . ) .

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F RT-PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C-control, M-Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).


A Matrin3 crosslinking in Matrin3 ‐ regulated pre ‐ mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3 ′ ss and 5 ′ ss , respectively , the upstream flanking exon and 500 nucleotides downstream of its 5 ′ ss and the downstream flanking exon with 500 nucleotides upstream of its 5 ′ ss . The iCLIP tags were mapped onto silenced ASE ( blue , n = 421 ) , enhanced ASE ( red , n = 187 ) and to control ASE ( grey , n = 16 , 242 ) , and percentage of occupancy is plotted .

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A Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.


B Same as in ( A ) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB / nPTB ‐ regulated ASE , silenced ASE ( blue , n = 729 ) , enhanced ( red , n = 820 ) and control ASE ( grey , n = 14 , 599 ) .

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B Same as in (A) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB/nPTB‐regulated ASE, silenced ASE (blue, n = 729), enhanced (red, n = 820) and control ASE (grey, n = 14,599).


C Matrin3 crosslinked nucleotides mapped onto PTB ‐ regulated ASE .

C Matrin3 crosslinked nucleotides mapped onto PTB‐regulated ASE.


D PTB crosslinked nucleotides mapped onto Matrin3 ‐ regulated ASE .

D PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.


B Western blot of the overexpressed Matrin3 and its mutants probed with anti ‐ FLAG and anti ‐ actin antibodies .

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B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.


C , D RT - PCR analysis using primers specific to the ST7 exon 11 ( C ) and ABI2 exon 8 ( D ) splicing reporters , respectively , of samples where FLAG ‐ tagged Matrin3 and its mutants have been overexpressed . Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion . * P 0 . 01 compared with control sample ( − ) .

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C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).


E Western blot of overexpressed FLAG ‐ tagged Matrin3 and PTB , in control siRNA and PTB / nPTB siRNA ‐ transfected cells , probed with anti ‐ FLAG to detect overexpressed proteins , anti ‐ PTB to detect the knockdown levels and anti ‐ actin to confirm protein loading . A titration of control sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % and lane 4 - 100 % ) is also included to assess the levels of knockdown . The band present above the PTB doublet in lane 6 is FLAG ‐ tagged PTB .

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E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.


F , G RT - PCR analysis using primers specific to the ST7 exon 11 ( F ) and ABI2 exon 8 ( G ) splicing reporters , respectively , of samples where Matrin3 or PTB expression vectors were co ‐ transfected , in control siRNA and PTB / nPTB siRNA ‐ transfected cells . Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion . * P 0 . 01 when compared with control sample in each condition ( ‐ ) .

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F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).


C . Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP - Cwh43 , GFP - Cwh43 - k696fs or GFP - Cwh43 - Leu533ter fusion proteins . To label the ER , HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP - calreticulin fusion protein containing an ER retention signal ( RFP - calreticulin - KDEL ) . To label the Golgi apparatus , cells were immunostained for golgin - 97 ( red ) . When compared with wild type GFP - Cwh43 , GFP - Cwh43 - k696fs and GFP - Cwh43 - Leu533 showed decreased association with the Golgi apparatus ( but not the ER ) and were diffusely distributed throughout the cytoplasm . Scale bar is approximately 5 µm .

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C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm.


D . Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout ( KO ) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced . Cells were transfected to overexpress a control GFP plasmid , a plasmid encoding human wild type Cwh43 with GFP fused to the N - terminus , or a plasmid encoding human CWH43 harboring the iNPH - associated mutation ( Lys696AsnfsTer23 ) with GFP fused to the N - terminus . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein .

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D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell . Cilia were visualized using an antibody for acetylated alpha tubulin ( green ) . Cwh43 immunoreactivity was visualized using a specific anti - Cwh43 antibody ( red ) . Scale bar ( left column ) is approximately 4 µm . Images in the column on the right represent a Z - stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell . Scale bar ( right column ) is approximately 5 µm .

Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.


A . Scatter plot comparing ventricular volume from CWH43WT / WT ( wild type , WT ) , heterozygous CWH43WT / M533 , homozygous CWH43M533 / M533 and CWH43M533 / A530 mice at 6 months . Ventricular volume was calculated from T2 - weighted MR images of the mouse brain using a custom automated computer algorithm . Horizontal bars indicate the mean of the measurements in each column . Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t - test ( P = 0 . 0015 for CWH43WT / M533 ; P = 0 . 0014 for CWH43M533 / M533 ; P = 0 . 006 for CWH43M533 / A530 ) .

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A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530).


D . Box plot showing rotarod performance data for CWH43WT / WT , CWH43WT / M533 , CWH43M533 / M533 and CWH43M533 / A530 mice at 7 months of age . Data shown are the mean , 1st quartile , 3rd quartile , minimum and maximum for each group of mice . Statistical significance was determined using the unpaired t - test . When compared to wild type CWH43WT / WT mice ( n = 10 ) , balance time on the rotarod was decreased significantly among heterozygous CWH43WT / M533 mice ( * P = 0 . 04 , n = 8 ) , homozygous CWH43M533 / M533 mice ( * P = 0 . 03 , n = 7 ) , and compound heterozygous CWH43M533 / A530 mice ( * P = 0 . 03 , n = 4 ) .

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D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).


Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533 / M533 mice . Scale bar is approximately 40 µm . The graph on the right quantifies the data from the electron micrographs on the left . Data shown are the mean ± SEM . * = P = 0 . 004 , n = 4 , unpaired t - test .

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Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533/M533 mice. Scale bar is approximately 40 µm. The graph on the right quantifies the data from the electron micrographs on the left. Data shown are the mean ± SEM. * = P=0.004, n = 4, unpaired t-test.


Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type , CWH43M533 / M533 and CWH43WT / M533mouse brain or kidney . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein .

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Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT / WT , CWH43M533 / M533 and CWH43M533 / A530 mice . Arrowheads point to apical surfaces of ependymal and choroid plexus cells . Nuclei are counterstained using DAPI ( blue ) . Scale bar is approximately 5 µm .

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Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.


D , E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation ( D ) , and for molecules involved in V ( D ) J recombination and TCR signaling ( E ) . In D and E , dot color represents the scaled average expression of the specified gene across the various clusters , and dot size indicates the percentage of cells expressing the specified gene .

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D, E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation (D), and for molecules involved in V(D)J recombination and TCR signaling (E). In D and E, dot color represents the scaled average expression of the specified gene across the various clusters, and dot size indicates the percentage of cells expressing the specified gene.


C UMAP plots colored according to the expression level of the specified genes .

C UMAP plots colored according to the expression level of the specified genes.


D Dot plot showing the expression level of genes belonging to the γδ TCR - induced signature across the cell subsets corresponding to Dataset 2 . Based on the gene set that was specifically upregulated during the DPsmall → DPCD69 + / SP transition ( see Fig EV3B ) , it allowed to subdivide the γδ TCR - induced signature into a ' γδ TCR only ' signature and a ' αβ and γδ TCR shared '

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D Dot plot showing the expression level of genes belonging to the γδ TCR-induced signature across the cell subsets corresponding to Dataset 2. Based on the gene set that was specifically upregulated during the DPsmall → DPCD69+/SP transition (see Fig EV3B), it allowed to subdivide the γδ TCR-induced signature into a 'γδ TCR only' signature and a 'αβ and γδ TCR shared' signature


A , B UMAP plots of Dataset 3 which contains five WT ( DN3a , DN3b γδ + , DN4 γδ + , DN3b γδ - and DN4 γδ - ) and two Lat - / - ( DN3 γδ - and DN3 γδint ) scRNAseq datasets . The UMAP plot is colored according to sorted samples ( A ) , and to expression levels of the specified genes ( B ) .

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A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B).


A , B , C AUCell scores for the γδ TCR - induced signature ( A ) , γδ TCR only signature ( B ) and αβ and γδ TCR shared signature ( C ) are displayed on UMAP ( left ) and violin ( right ) plots of the 7 sorted subsets corresponding to Dataset 3 . As expected , AUCell scores for the 3 signatures were very low for DN3a cells , low for WT DN3b γδ - and WT DN4 γδ - cells , and high for WT DN3b γδ + and WT DN4 γδ + cells .

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A,B,C AUCell scores for the γδ TCR-induced signature (A), γδ TCR only signature (B) and αβ and γδ TCR shared signature (C) are displayed on UMAP (left) and violin (right) plots of the 7 sorted subsets corresponding to Dataset 3. As expected, AUCell scores for the 3 signatures were very low for DN3a cells, low for WT DN3b γδ- and WT DN4 γδ- cells, and high for WT DN3b γδ+ and WT DN4 γδ+ cells.


B Bar plot showing the percentage of cells in the G0 / G1 ( red bars ) , S ( blue bars ) and G2 / M ( green bars ) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3 .

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B Bar plot showing the percentage of cells in the G0/G1 (red bars), S (blue bars) and G2/M (green bars) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3.


, C . siRNA - mediated knockdown of EXOSC10 ( EXO10 ) , but not of DIS3 and DIS3L , significantly increased generation of poly - GA in HeLa cells expressing ( G4C2 ) 80 repeats . ctrl " indicates a control vector which lacks the G4C2 repeat but still contains the 5 ' flanking region and 3xTAG .

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, C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG.


. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT - qPCR .

. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR.


E , F . Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing ( G4C2 ) 80 repeats visualized by in situ hybridization targeting sense RNA foci . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments . G . Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing ( G4C2 ) 80 repeats . Each dot represents average cellular RNA foci intensity ( Arbitrary unit ) of RNA foci - positive cells in each randomly taken image . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments .

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E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.


A , B . Dose - dependent suppression of poly - GA by exogenous EXOSC10 upon EXOSC10 knockdown .

A, B. Dose-dependent suppression of poly-GA by exogenous EXOSC10 upon EXOSC10 knockdown.


C . Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown . Concentration of transfected EXOSC10 plasmid was 0 . 2 ng / μl in 1 ml medium .

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C. Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown. Concentration of transfected EXOSC10 plasmid was 0.2 ng/μl in 1 ml medium.


D . Overexpression of EXOSC10 decreases G4C2 repeat RNA .

D. Overexpression of EXOSC10 decreases G4C2 repeat RNA.


A , B . Under transcription inhibition with Actinomycin D , cells with EXOSC10 knockdown ( EXO10 KD ) showed more stable expression of the repeat RNA compared to cells with control knockdown with non - targeting siRNA ( Ct KD ) .

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A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD).


C , D . Degradation of FAM - labeled synthetic ( G4C2 ) 8 repeat RNA with recombinant human EXOSC10 ( hEXO10 ) ( Upper left ) . 3 ' blocked FAM - labeled G4C2 repeat RNA resisting 3 ' exoribonuclease activity is not degraded by recombinant EXOSC10 ( Lower left ) . Lack of recombinant EXOSC10 ( Upper right ) or Mg2 + ion which is essential co - factor of EXOSC10 ( Lower right ) abolished the RNA degradation activity . 3 or 4 independent experiments . Quantification of the top bands are shown in ( D ) .

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C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).


E , F . Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 ( Left ) . Without recombinant EXOSC10 , no substrate degradation was observed ( Right ) . 5 or 6 independent experiments . Quantification is shown in ( F ) .

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E, F. Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 (Left). Without recombinant EXOSC10, no substrate degradation was observed (Right). 5 or 6 independent experiments. Quantification is shown in (F).


A . In situ hybridization . G4C2 RNA foci were detected in a C9 patient - derived fibroblast but not in a control - derived fibroblast with non - targeting siRNA treatment ( left panels , control / si Ct . middle panels , C9 / si Ct ) . Knockdown of EXSOC10 increased RNA foci ( right panels , C9 / siEXOSC10 # 5 ) . Nuclei were stained with DAPI ( blue ) . White arrows indicate nuclear RNA foci . The green arrows show cytoplasmic RNA foci .

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A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci.


B . Quantification of % cell with G4C2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . C . Quantification of the number of G4C2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . D . Quantification of % cell with C4G2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . E . Quantification of the number of C4G2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci .

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B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.


F , G . Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT - qPCR . 10 independent knockdown experiments ( Case1 N = 4 , Case2 N = 4 , Case3 N = 2 ) were performed .

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F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.


A , B , C . Double knockdown of EXOSC10 and DIS3 significantly increased poly - GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing ( G4C2 ) 80 repeats .

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A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats.


D . Overexpression of DIS3 compensated EXOSC10 knockdown - induced repeat RNA accumulation .

D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation.


E , F . Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown - induced GA increase .

E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase.


C - F . HA - tagged poly - GA , poly - GP , poly - GR or poly - PR ( red ) and EXOSC10 ( green ) were immunostained with anti - HA and anti - EXOSC10 antibodies . Nuclei were stained with DAPI ( blue ) .

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C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue).


G . EXOSC10 ( green ) were immunostained with anti - EXOSC10 antibody in cells transfected with control mock plasmid . Nuclei were stained with DAPI ( blue ) .

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G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue).


H , I . Quantification of the frequency of cells with mislocalized EXOSC10 . Only DPR - positive cells were quantified ( except mock transfected cells expressing no DPR . ( Total cell counts : ( figure H ) mock 300 cells , GA 112 cells , GP 69 cells , GR 53 cells , ( figure I ) mock 445 cells , PR 159 cells ) .

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H, I. Quantification of the frequency of cells with mislocalized EXOSC10. Only DPR-positive cells were quantified (except mock transfected cells expressing no DPR. (Total cell counts: (figure H) mock 300 cells, GA 112 cells, GP 69 cells, GR 53 cells, (figure I) mock 445 cells, PR 159 cells).


B . Immunoblot validation of successful fractionation using antibodies against HDAC1 ( nuclear marker ) and GAPDH ( cytoplasmic marker ) .

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B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker).


C . Co - immunoprecipitation of GFP - DPR interacting proteins in cytoplasmic fraction using GFP - trap beads . Immunoprecipitants were served for immunoblot with anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . D . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " . E . Co - immunoprecipitation of GFP - DPR interacting proteins in nuclear fraction using GFP - trap beads followed by immunoblot using anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . F . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " .

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C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input".


B , C . Increased expression of 3 ' extended snoRA48 precursor upon EXOSC10 knockdown ( B ) or GFP - poly - GR177 / GFP - poly - PR166 expression ( C ) on RT - qPCR . D , E . Increased expression of 3 ' extended snoRA68 precursor upon EXOSC10 knockdown ( D ) or GFP - poly - GR177 / GFP - poly - PR166 expressions ( E ) on RT - qPCR .

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B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR.


F . RT - qPCR analysis showed increased cellular G4C2 repeat RNA expression from co - transfected EF1 ( G4C2 ) 80 repeat construct in the presence of GFP , GFP - poly - GR177 or GFP - poly - PR166 .

F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166.


( a - f ) HepG2 cells were treated with bovine serum albumin ( Con ) , 500 μM PA or 100 nM rapamycin ( Rap ) for 9 h and subjected to following analyses . ( a , c - f ) Cells were stained with ubiquitin ( Ub ) , p62 , LC3 and LAMP1 antibodies and DAPI ( blue ) . Boxed areas are magnified in right - most panels ( c , d ) . Co - localization between LAMP1 and LC3 staining ( e ) was quantified ( f ; n = 3 ) .

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

(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).


( b ) Cells were subjected to serial protein extraction ( solubility fractionation ) with indicated concentration of Triton X - 100 ( TX100 ) or sodium dodecyl sulphate ( SDS ) and analysed by immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O 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) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulphate (SDS) and analysed by immunoblotting with indicated antibodies.


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

(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).


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


( g , h ) HepG2 cells stably transduced with mCherry ( mCh ) - GFP - LC3 - expressing retroviruses were treated with Con , Rap or PA for 9 h and examined under a live confocal microscope ( g ) . Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out . Number of autolysosomes was quantified ( h ; n = 7 ) . Scale bar , 5 μm . All data are shown as mean ± s . e . m . * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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

(g,h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 h and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h; n=7). Scale bar, 5 μm. All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


HepG2 cells were treated with bovine serum albumin ( Con or ( − ) ) , PA ( 500 μM ) , PA + verapamil ( Ver , 50 μM ) or PA + nicardipine ( Nic , 100 μM ) for 9 h . ( a - d ) After each treatment , cells were loaded with a calcium indicator X - Rhod - 1 - AM ( a , b ) or Fura - 2 - AM ( c , d ) . Calcium levels were visualized by laser confocal microscopy ( a ) or by dual fluorescent microscopy ( c , 340 / 380 nm ratio image ) and quantified ( b , d ; n = 8 and 30 , respectively ) .

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

HepG2 cells were treated with bovine serum albumin (Con or (−)), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 9 h. (a-d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a,b) or Fura-2-AM (c,d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b,d; n=8 and 30, respectively).


( e - h ) Cells with indicated treatments were subjected to solubility fractionation . 1 % Triton X - 100 - insoluble fractions were dissolved in 2 % SDS , analysed by immunoblotting ( e , f ) and quantified ( g , h ; n = 3 ) .

(e-h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analysed by immunoblotting (e,f) and quantified (g,h; n=3).


( i - l ) Cells with indicated treatments were subjected to immunostaining with indicated antibodies ( i , k ) . DNA was stained with DAPI ( blue ) . Amount of aggregated proteins was quantified ( j ; n = 10 ) . Co - localization between LAMP1 and LC3 was quantified ( l ; n = 4 ) . Boxed areas in fluorescence images are magnified in right - most panels ( i , k ) . Scale bar , 10 μm ( a ) , 20 μm ( c ) , 5 μm ( i , k ) . All data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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

(i-l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i,k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j; n=10). Co-localization between LAMP1 and LC3 was quantified (l; n=4). Boxed areas in fluorescence images are magnified in right-most panels (i,k). Scale bar, 10 μm (a), 20 μm (c), 5 μm (i,k). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg body weight , i . p . , n = 3 ) for 10 days . Low - fat diet ( LFD ) - kept mice ( n = 5 ) of same age were used as a negative control . ( a ) Body weight was daily monitored during injection period . ( b ) Daily food consumption was measured during injection period .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet (LFD)-kept mice (n=5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period.


( c ) Livers were collected from indicated mice and photographed . ( d , e ) Total liver mass ( d ) were measured from indicated mice .

O O O O O 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) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) were measured from indicated mice.


( c ) Livers were collected from indicated mice and photographed . ( d , e ) Total liver mass ( d ) and total epididymal white adipose tissue ( eWAT ) mass ( e ) were measured from indicated mice .

O O O O O O O O O O O O O O O O O O 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) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) and total epididymal white adipose tissue (eWAT) mass (e) were measured from indicated mice.


( f - h ) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription - PCR .

(f-h) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription-PCR.


( i , j ) Liver sections were analysed by hematoxylin and eosin ( HE , upper panels ) and Oil Red O ( ORO , lower panels ) staining ( i ) . ORO densities were quantified ( j ) .

(i,j) Liver sections were analysed by hematoxylin and eosin (HE, upper panels) and Oil Red O (ORO, lower panels) staining (i). ORO densities were quantified (j).


( k , l ) Calcium - induced CaMKII autophosphorylation in livers was analysed by immunoblotting ( k ) and quantified ( l ) .

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

(k,l) Calcium-induced CaMKII autophosphorylation in livers was analysed by immunoblotting (k) and quantified (l).


( m ) Primary hepatocytes from 2 - month - old C57BL / 6 mice kept on LFD were treated with bovine serum albumin ( Con ) , PA ( 500 μM ) , PA + verapamil ( Ver , 50 μM ) or PA + nicardipine ( Nic , 100 μM ) for 12 h . After each treatment , cells were loaded with a calcium indicator Fura - 2 - AM . Calcium levels were visualized by dual fluorescent microscopy at 340 / 380 nm , and ratio of fluorescence intensities of Fura - 2 - AM at 340 nm over 380 nm was quantified ( n = 17 ) . Scale bar , 1 cm ( c ) , 200 μm ( i ) . All data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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

(m) Primary hepatocytes from 2-month-old C57BL/6 mice kept on LFD were treated with bovine serum albumin (Con), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 12 h. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n=17). Scale bar, 1 cm (c), 200 μm (i). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg body weight , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice ( n = 5 ) of same age were used as a negative control . ( a - d ) Livers were subjected to solubility fractionation . 1 % Triton X - 100 - soluble ( a ) and - insoluble ( b ) fractions were analysed by immunoblotting ( a , b ) and quantified ( c , d ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a-d) Livers were subjected to solubility fractionation. 1% Triton X-100-soluble (a) and -insoluble (b) fractions were analysed by immunoblotting (a,b) and quantified (c,d).


( e , f ) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining ( e ) . p62 - positive areas were quantified ( f ) . Scale bar , 200 μm ( e ) . All data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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

(e,f) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining (e). p62-positive areas were quantified (f). Scale bar, 200 μm (e). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg body weight , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice ( n = 5 ) of same age were used as a negative control . ( a , b ) Levels of LC3 - II from 1 % Triton X - 100 - insoluble fraction of livers were analysed by immunoblotting ( a ) and quantified ( b ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a,b) Levels of LC3-II from 1% Triton X-100-insoluble fraction of livers were analysed by immunoblotting (a) and quantified (b).


( c , d ) Frozen sections of indicated livers were subjected to LC3 / LAMP1 immunostaining and DAPI counterstaining ( c ) . Co - localization between LC3 and LAMP1 was quantified ( d ) . Boxed areas in fluorescence images are magnified in right - most panels ( c ) . Scale bar , 5 μm ( c ) . All data are shown as mean ± s . e . m . * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,d) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining (c). Co-localization between LC3 and LAMP1 was quantified (d). Boxed areas in fluorescence images are magnified in right-most panels (c). Scale bar, 5 μm (c). All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg body weight , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice of same age ( n = 5 ) were used as a negative control . ( a , b ) Liver sections were subjected to F4 / 80 immunostaining , which visualizes macrophage infiltration and hematoxylin counterstaining ( a ) . F4 / 80 - positive areas were quantified ( b ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice of same age (n=5) were used as a negative control. (a,b) Liver sections were subjected to F4/80 immunostaining, which visualizes macrophage infiltration and hematoxylin counterstaining (a). F4/80-positive areas were quantified (b).


( c - f ) Glucose tolerance tests ( GTT , c , d ) and insulin tolerance tests ( ITT , e , f ) were conducted using indicated mice ( c , e ) . Area under the curve ( AUC ) was quantified from GTT and ITT data ( d , f ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Glucose tolerance tests (GTT, c,d) and insulin tolerance tests (ITT, e,f) were conducted using indicated mice (c,e). Area under the curve (AUC) was quantified from GTT and ITT data (d,f).


( g ) Serum insulin levels were measured from indicated mice before ( Basal ) and 10 min after ( Glucose stimulated ) glucose injection ( n = 4 ) .

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

(g) Serum insulin levels were measured from indicated mice before (Basal) and 10 min after (Glucose stimulated) glucose injection (n=4).


( h , i ) Pancreas sections were analysed by hematoxylin and eosin ( HE ) staining ( h ) . Islet areas were quantified ( i ; n = 4 ) . Scale bar , 200 μm ( a ) , 100 μm ( h ) . All data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - test ) .

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

(h,i) Pancreas sections were analysed by hematoxylin and eosin (HE) staining (h). Islet areas were quantified (i; n=4). Scale bar, 200 μm (a), 100 μm (h). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test).


( a - g ) Four - month - old C57BL / 6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg body weight , i . p . , n = 4 ) for 10 days . Systolic blood pressure ( a ) , LV wall mass ( b ) , diastolic LV volume ( c ) , systolic LV volume ( d ) , ejection fraction ( e , % EF ) , stroke volume ( f , SV ) and cardiac output ( g , CO ) were analysed by tail - cuff method ( a ) or echocardiography ( b - g ) . All data are shown as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ) .

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

(a-g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=4) for 10 days. Systolic blood pressure (a), LV wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV) and cardiac output (g, CO) were analysed by tail-cuff method (a) or echocardiography (b-g). All data are shown as mean±s.e.m. *P0.05 (Student's t-test).


( A ) Western blotting of AXL and OPCML protein levels in AXL - expressing , OPCML - null SKOV3 and PEA1 cell lines transduced with OPCML " O " or control Empty vector " E "

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

(A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E"


( B ) Mammalian 2 - Hybrid assay between OPCML and AXL Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mammalian 2-Hybrid assay between OPCML and AXL Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( C ) Western blotting of the OPCML - GST pull down . Input : 1 / 20 of SKOV3 - Empty whole cell

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) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate


( D ) Western blotting of the anti - AXL immunoprecipitation . Input : 1 / 100 of SKOV3 - OPCML whole cell lysate . IP : Immunoprecipitated protein , IB : Immunoblotted

O O O O O O O O O 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) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( E ) Immunostaining of OPCML ( red ) and AXL ( green ) in SKOV3 - OPCML and PEA1 - OPCML cells . Scale bar = 10μm Data i M , ( E ) was performed once due to the limited numbers of primary cells ;

O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;


( G ) FRET efficiency for AXL - OPCML interaction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " , using AXL - AXL FRET as a positive control . Cells were labeled with Anti - Axl rabbit ( conjugated to donor probe ) and Anti - OPCML mouse ( conjugated to acceptor probe ) , or Anti - Axl mouse ( conjugated to acceptor probe ) and Anti - Axl rabbit ( conjugated to donor probe ) Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

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

(G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( H ) PLA of AXL - OPCML ( red ) in SKOV3 and PEA1 cells transduced with empty vector " E " or OPCML " O " . Scale bar =

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

(H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μm


Primary ovarian tumour cells were characterised for AXL and OPCML levels by ( I ) Western blottin PAX8 was used to identify ovarian cancer

Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cells


Primary ovarian tumour cells were characterised for AXL and OPCML levels b ( J ) Immunostaining . PAX8 was used to identify ovarian cancer

Primary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells


( K ) Quantitation of AXL - OPCML PLA in primary cells Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

(K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( L ) Western blotting of membrane fractionation of SKOV3 - Empty cells : total cell lysate " T " , liquid - disordered soluble fraction " S " , detergent resistant membrane fraction " R " . Caveolin - 1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction , E : SKOV3 - Empty , O : SKOV3 -

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

(L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML


( M ) Densitometry of AXL band intensities in ( J ) in S and R , normalized to AXL intensity in input . Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

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

(M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


SKOV3 - OPCML cells stimulated with Gas6 over a 12 hour time course and ( A ) stained with DAPI ( cyan ) , Anti - OPCML ( green ) and Anti - AXL ( red ) antibodies ( scale bar = 10μm )

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

SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm)


( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


d ; ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n =

O O O O O 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; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3


( E ) Quantitation OPCML - AXL PLA interaction in primary ovarian tumour cells , n = 1 Data i M , ( E ) was performed once due to the limited numbers of primary cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( F ) Co - immunoprecipitation with anti - AXL antibody in cells stimulated with Gas6 as indicated . Input : 1 / 50 of whole cell lysate from SKOV3 - OPCML cells . IP : Immunoprecipitated protein , IB : Immunoblotted

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

(F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( G ) Co - immunoprecipitation with anti - pAXL antibody in SKOV3 - OPCML cells stimulated with

(G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6


( H ) OPCML - GST pull down assay in SKOV3 - Empty cells stimulated with

(H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6


( I ) Membrane fractionation in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6

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

(I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( K ) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

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

(K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( L ) Diffusion Co - efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

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

(L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( M ) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

(M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( N ) FRET Efficiency in SKOV3 - OPCML " O " cells stimulated with Gas6 , and labeled with Anti - Axl ( donor probe ) and Anti - OPCML ( acceptor probe ) Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

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

(N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( O ) Western blotting of the " S " and " R " membrane fractions in SKOV3 - Empty " E " and OCPML " O " cells stimulated with Gas6 for 3 hours , for Gas6 , AXL and pAXL . Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ±

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

(O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM


( A ) Filipin staining of SKOV3 - OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0 , 30 minutes , and 12 hours in serum free ( SF )

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

(A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium


( B ) Western blotting of SKOV3 - OPCML cells treated with MBCD and separated into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6

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

(B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( C ) Ratio of AXL band intensity in " R " or pAXL band intensity in " S " relative to total AXL " T " from SKOV3 - OPCML " O " in ( B )

O O O O O O O O O O O O O O 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) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B)


( D ) MBCD treatment of SKOV3 - EMPTY and SKOV3 - OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot . GAPDH was used as loading

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

(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control


( E , F ) Signalling kinetics are presented for pAXL and pERK in SKOV3 - EMPTY " E " and SKOV3 - OCPML " O " , treated with MBCD and no treatment control , normalised to GAPDH , from ( D )

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

(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)


( G ) PLA assay of AXL - OPCML interaction ( red ) upon Gas6 stimulation in both MBCD treated and non - treated SKOV3 - OPCML cells . Scale bar =

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

(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm


( B ) Western Blotting of SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells pre incubated with either no drug ( DMSO only control ) , 1 . 09 μM R428 , or 2 . 84 μM R428 for an hour and stimulated with Gas6 for the indicated times . GAPDH was used as loading

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

(B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control


( C ) Immunofluorescence staining of pERK ( red ) and OPCML ( magenta ) in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with R428 and stimulated with Gas6 for the indicted time points . Scale bars =

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

(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm


Bone marrow cells were polarized in the presence of 40 ng / ml GM - CSF and treated with 0 . 2 mM of the indicated compounds for 7 days . Gr1 - CD11b + cells were sorted and lysed for microarray . The hierarchical clustering was based on the different expression genes between BSA ( Con ) and oleate group .

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

Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group.


Signature genes involved in lipid metabolism , dendritic cell maturation , macrophage maturation , tumor associated macrophages ( TAMs ) phenotype , MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay . Data are expressed as mean ± SD from two to four independent experiments . Unpaired Student ' s two - tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups . * p < 0 . 05 ; * * p ≤ 0 .

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

Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01


Gr1 - CD11b + cells , polarized by the indicated treatment , were purified for mitochondrial respiration detection . The oxygen consumption rate ( OCR ) of these cells was monitored after the addition of oligomycin ( OA ) ( 1 µM ) , Carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA ) ( 0 . 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified .

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

Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


Forty µM etomoxir were added starting from day 0 of bone marrow polarization , followed by mitochondrial respiration assay by using the same amount of polarized Gr1 - CD11b + myeloid cells on day 7 . The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer .

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

Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer.


Exemplary plots of the proportion of CD206 + cells , the mean fluorescence intensity ( MFI ) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry .

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

Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry.


T cell proliferation assay was performed via co - culture with purified CD4 + T cells in the ratios of M ( myeloid cells ) : T ( T cells ) = 1 : 30 .

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

T cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30.


NO production from the co - culture supernatant was quantified by Griess reaction .

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

NO production from the co-culture supernatant was quantified by Griess reaction.


The oxygen consumption rate ( OCR ) of 1 × 105 purified Gr1 - CD11b + cells was monitored as described in Figure 2 . The basal OCR , basal ECAR ( extracellular acidification rate ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified .

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

The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.


The polarization state was evaluated via the expression of CD206 .

The polarization state was evaluated via the expression of CD206.


T cell proliferation assay was performed employing co - culture with purified CD4 + T cell in the variant ratios .

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

T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios.


The lipid droplets accumulation was determined by BODIPY staining . The percentage of divided cells and proliferation index were calculated .

The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated.


Nitric oxide ( NO ) concentration in the co - culture supernatant was quantified by Griess reaction .

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

Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.


Bone marrow - derived myeloid cells were treated with BSA - control ( Con ) or oleate in the presence or absence of 10 nM rapamycin ( Rapa ) . The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression .

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

Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.


Purified Gr1 - CD11b + myeloid cells were co - cultured with CD4 + T cells for proliferation assay . The supernatant was collected for nitric oxide ( NO ) detection .

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

Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.