( i ) Representative of immuno - histochemical of SLC7A11 YAP in HCC samples from . Scale bars , 50μm . ( j ) of immunohistochemical stainings shown in ( i ) revealed a positive correlation of YAP SLC7A11 expression ( N = 10 ) . Statistical significance was calculated using Pearson correlation .

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(i) Representative images of immuno-histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (j) Quantification of the immunohistochemical stainings shown in (i) revealed a positive correlation of YAP and SLC7A11 expression (N=10). Statistical significance was calculated using Pearson correlation analysis.


( a ) SLC7A11 decreased upon siRNA - mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment , Sorafenib promoted expression of ATF4 . HLE were transfected with siCtrl , siATF4 or siNRF2 treated with or without 6μM Sorafenib for 18 hours . expression of SLC7A11 , ATF4 NRF2 was determined by immunoblotting . GAPDH served as loading control . Results represent three independent experiments .

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(a) SLC7A11 protein levels decreased upon siRNA-mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment, and Sorafenib promoted the expression of ATF4. HLE cells were transfected with siCtrl, siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours. The expression of SLC7A11, ATF4 and NRF2 was determined by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.


( f ) Colony formation demonstrating that ferroptosis inhibitor Ferrostatin - 1 ( Fer ) reversed Sorafenib - induced in ATF4 - deficient HCC . HLE transfected with siCtrl or siATF4 were treated with Sorafenib ( 8μM ) or DMSO plus either DMSO or Ferrostatin - 1 ( Fer ; 5μM ) for 2 . Results represent three independent experiments .

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(f) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in ATF4-deficient HCC cells. HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib (8μM) or DMSO plus either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.


( g ) forced expression of SLC7A11 rescued induced by ATF4 ablation . HLE were transfected with a construct coding for SLC7A11 ( SLC7A11 OE ) or empty vector ( EV ) with siCtrl or siATF4 every other day . were then treated with either DMSO or 6μM Sorafenib for 2 . Colony formation was visualized by crystal violet . Results represent three independent experiments .

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(g) The forced expression of SLC7A11 rescued cell death induced by ATF4 ablation. HLE cells were transfected with a construct coding for SLC7A11 (SLC7A11 OE) or empty vector (EV) and with siCtrl or siATF4 every other day. Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks. Colony formation was visualized by crystal violet staining. Results represent three independent experiments.


( h ) Immunohistochemical of ATF4 in HCC adjacent non - neoplastic areas from . tissues showed a higher expression of ATF4 . Scale bar , 100μm . ( i ) of ATF4 - positive in non - samples showed that HCC tumors present higher ATF4 . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using unpaired t - . ( j ) Representative of immuno - histochemical of ATF4 SLC7A11 in HCC samples from . Scale bars , 50μm .

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(h) Immunohistochemical staining of ATF4 in HCC and adjacent non-neoplastic areas from patients. Tumor tissues showed a higher expression of ATF4. Scale bar, 100μm. (i) Quantification of ATF4-positive cells in tumor and non-tumor samples showed that HCC tumors present higher ATF4 levels. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using unpaired t-test. (j) Representative images of immuno-histochemical staining of ATF4 and SLC7A11 proteins in HCC samples from patients. Scale bars, 50μm.


( a ) YAP / TAZ deficiency repressed Sorafenib - induced expression of ATF4 , whereas ATF4 depletion had no effect on YAP / TAZ . HLE were transfected with siCtrl or siATF4 or siY / T treated with 6μM Sorafenib or not for 18 hours . ATF4 YAP / TAZ were analyzed by immunoblotting . GAPDH served as loading control . Results represent three independent experiments .

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(a) YAP/TAZ deficiency repressed Sorafenib-induced expression of ATF4, whereas ATF4 depletion had no effect on YAP/TAZ protein levels. HLE cells were transfected with siCtrl or siATF4 or siY/T and treated with 6μM Sorafenib or not for 18 hours. ATF4 and YAP/TAZ protein levels were analyzed by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.


( d ) fractionation revealed that high density , with it , lower YAP / TAZ activity reduced of ATF4 . HLE were seeded at different numbers , lysates of high density ( HD ) low density ( LD ) were fractionated into fractions analyzed for ATF4 YAP / TAZ by immunoblotting . GAPDH served as loading control for total lysate , Lamin A / C served as loading control for . Results represent three independent experiments .

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(d) Cellular fractionation revealed that high cell density, and with it, lower YAP/TAZ activity reduced the levels of nuclear ATF4. HLE cells were seeded at different cell numbers, and cell lysates of high cell density cells (HD) and low cell density (LD) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP/TAZ by immunoblotting. GAPDH served as loading control for total lysate and cytoplasmic proteins, Lamin A/C served as loading control for nuclear proteins. Results represent three independent experiments.


( h ) Representative of immuno - histochemical of ATF4 YAP in HCC samples from . Scale bars , 50μm . ( i ) of immunohistochemical in ( j ) showed a positive correlation of ATF4 YAP expression ( N = 18 ) . Statistical significance was calculated using Pearson correlation .

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(h) Representative images of immuno-histochemical staining of ATF4 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (i) Quantification of the immunohistochemical staining in (j) showed a positive correlation of ATF4 and YAP expression (N=18). Statistical significance was calculated using Pearson correlation analysis.


( b ) Quantitative RT - PCR verified that depletion of YAP / TAZ or ATF4 declined expression of SLC7A11 CHAC1 . HLE were transfected with siCtrl , siY / T or siATF4 cultured with 6μM Sorafenib for 18 hours . Quantitative RT - PCR was conducted to determine SLC7A11 CHAC1 mRNA . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using Two - way ANOVA . Results represent 3 independent experiments .

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(b) Quantitative RT-PCR analysis verified that depletion of YAP/TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siY/T or siATF4 and cultured with 6μM Sorafenib for 18 hours. Quantitative RT-PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels. Data are shown as mean ± standard deviation (SD).Statistical significance was calculated using Two-way ANOVA. Results represent 3 independent experiments.


( e ) Binding of YAP TAZ to fragments containing AARE binding motif in SLC7A11 promoter . ChIP was performed on HLE lysate with antibodies against YAP TAZ rabbit IgG as control . fragments were amplified using primers specific for AARE binding motif in SLC7A11 promoter region . non - coding region NC10 served as negative control , bona fide TEAD target genes CYR61 ANKRD1 served as positive controls . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(e) Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region. The non-coding region NC10 served as negative control, and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


( g ) YAP / TAZ bind to fragments containing AARE binding motif within SLC7A11 promoter via ATF4 . HLE were cultured with 6μM Sorafenib for 18 hours before harvest . In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4 , rabbit IgG was used as control . - immunocomplexes were eluted in a 2nd round ChIP antibody against YAP / TAZ was used to precipitate fragments which were then amplified analyzed by quantitative PCR for AARE motif in SLC7A11 promoter . NC10 served as negative PCR control . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(g) YAP/TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4. HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest. In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4, rabbit IgG was used as control. DNA-protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP/TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter. NC10 served as negative PCR control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


Combination of YAP / TAZ deficiency Sorafenib treatment suppressed growth in a HCC xenograft model . SNU398 - shLuc or SNU398 - shYAP / TAZ ( shY / T ) were transplanted into flanks of immunodeficient NSG mice . Once tumors were palpable , mice were treated with 20mg / kg Sorafenib or vehicle control , sizes were measured twice a week ( b ) . weights were also recorded after sacrifice of mice Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA . Mouse numbers of shLuc + Vehicle , shY / T + Vechicle shY / T + Srf were 4 per cohort , mouse numbers of shLuc + Srf were 3 per cohort .

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Combination of YAP/TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model. SNU398-shLuc or SNU398-shYAP/TAZ (shY/T) cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with 20mg/kg Sorafenib or vehicle control, and tumor sizes were measured twice a week (b). Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers of shLuc + Vehicle, shY/T + Vechicle and shY/T + Srf were 4 per cohort, mouse numbers of shLuc + Srf were 3 per cohort.


( e ) Pharmacological inhibition of glutathione synthesis function sensitizes HCC tumors to Sorafenib therapy . SNU398 were transplanted into flanks of immunodeficient NSG mice . Once tumors were palpable , mice were treated with vehicle control or with 20mg / kg Sorafenib alone , or with 20mg / kg Sorafenib 20mM BSO in drinking water or 120mg / kg SSA per os . sizes were measured twice a week . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA . Mouse numbers were 8 for experimental cohorts Vehicle , Srf , Srf + SSA 7 for Srf + BSO .

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(e) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy. SNU398 cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with vehicle control or with 20mg/kg Sorafenib alone, or with 20mg/kg Sorafenib and 20mM BSO in the drinking water or 120mg/kg SSA per os. Tumor sizes were measured twice a week. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers were 8 for the experimental cohorts Vehicle, Srf, and Srf + SSA and 7 for Srf + BSO.


( g ) of 4 - HNE - positive of immunohistochemical stainings described in ( f ) . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Each data point represents average of 3 technical replicates of one histological section , numbers of samples are given in ( e ) .

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(g) Quantification of 4-HNE-positive cells of the immunohistochemical stainings described in (f). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Each data point represents the average of 3 technical replicates of one histological section, the numbers of tumor samples are given in (e).


B ) Electrophoretic mobility shift ( EMSA ) to examine binding affinities of GST - KNL2518 - 560 fragment for CENP - A or H3 nucleosome . molar of KNL2518 - 560 against nucleosomes are indicated .

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B) Electrophoretic mobility shift assay (EMSA) to examine binding affinities of the GST-KNL2518-560 fragment for the CENP-A or H3 nucleosome. The molar ratio of KNL2518-560 against nucleosomes are indicated.


D ) - F ) EMSA to examine binding affinities of GST - KNL2 fragment for CENP - A nucleosome : D , GST - KNL2347 - 560 ; E , GST - KNL2456 - 560 ; F , GST - KNL2456 - 516 ( CC - upstream ) .

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D)-F) EMSA to examine binding affinities of the GST-KNL2 fragment for the CENP-A nucleosome: D, GST-KNL2347-560; E, GST-KNL2456-560; F, GST-KNL2456-516 (CC-upstream).


B ) EMSA to examine contribution of ggKNL2 R527 residue to CENP - A nucleosome binding .

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B) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP-A nucleosome binding.


D ) Expression of WT or R527A mutant GFP - KNL2 in KNL2 conditional knock out chicken DT40 ( cKO - KNL2 ) . Expression of full - KNL2 WT was conditionally turned off by tetracycline ( tet ) addition in cKO - KNL2 ( None ) . GFP - fused KNL2 WT or R527A mutant was stably expressed in cKO - KNL2 . These were cultured in presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control .

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D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analyses of GFP - fused KNL2 WT or R527A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm .

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E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.


F ) Growth of cKO - KNL2 expressing GFP - fused KNL2 WT or R527A mutant . upper panel shows examined numbers at indicated time after tet addition ( + tet : KNL2 OFF ) . numbers were normalized to those at 0 h for each plotted as relative number . lower panel shows examined untreated ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 ( None ) were also examined .

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F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


B ) EMSA to examine contribution of F535 W536 residues in ggKNL2 to CENP - A nucleosome binding .

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B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.


D ) Expression of GFP - KNL2 WT or CENP - A C - terminal tail binding mutants in cKO - KNL2 . Expression of full - KNL2 WT was conditionally turned off by tetracycline ( tet ) addition to cKO - KNL2 ( None ) . GFP - fused KNL2 WT , F535A , W536A or F535A / W536A was stably expressed in cKO - KNL2 . These were cultured in presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control .

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D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analyses of GFP - fused KNL2 WT F535A , W536A or F535A / W536A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm .

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E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.


F ) Growth of cKO - KNL2 expressing GFP - fused KNL2 WT or each CENP - A C - terminal tail binding mutant . upper panel shows examined numbers at indicated time after tet addition ( + tet : KNL2 OFF ) . numbers were normalized to those at 0 h for each plotted as relative number . lower panel shows examined untreated ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 ( None ) were also examined .

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F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


A ) Immunostaining with anti - Mis18α antibody in AID - KNL2 knockout expressing GFP fused KNL2 wild type or each KNL2 mutant in presence of IAA for 15 h . Clear colocalization of KNL2 signals with Mis18α was observed ( Insets ) . was stained with DAPI . Scale bar indicates 10 µm .

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A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.


B ) AID - KNL2 knockout expressing GFP fused each KNL2 mutant SNAP - tagged CENP - A were used . After addition of IAA , SNAP - CENP - A signals were visualized by TMR - Star . CENP - T was stained as a kinetochore marker . Representative were presented . Mitotic centromere localization of each KNL2 mutant was shown as a merged image ( Insets ) : SNAP - CENP - A ( red ) each KNL2 mutant ( green ) . was stained with DAPI . Scale bar indicates 10 µm .

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B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.


A ) KNL2 ( red ) localization in CENP - C conditional knockout chicken DT40 ( cKO - CENP - C ) with or without CENP - C expression . was stained with DAPI ( blue ) . CENP - T was stained as a kinetochore marker ( blue ) . Scale bar indicates 10 μm .

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

A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.


B ) KNL2 intensities were evaluated in six before after CENP - C knock out . KNL2 signal intensities on mitotic centromeres ( 14 - 90 centromeres ) in each were plotted with average standard deviation . of KNL2 intensities between before after CENP - C knock out was estimated . KNL2 intensities were reduced by ~ 55 % , when CENP - C was knocked out .

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

B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.


C ) Competitive pull - down assays for CENP - A nucleosome binding of ggKNL2 ggCENP - C . A of GST - KNL2456 - 560 with CENP - A nucleosome ( CA - nuc ) was incubated in absence or presence of MBP - CENP - C619 - 690 ( right panel ) or phosphorylated MBP - CENP - C619 - 690 ( middle panel ) pulled down by GST affinity . of CENP - C619 - 690 was increased from 0 μM to 1 μM ( lanes 3 - 7 ) . of band intensities of pull - downed histones ( * ) to GST - KNL2456 - 560 in each lane ( lanes 3 - 7 ) was estimated presented in bar graph ( left panel ) . Data are mean with standard deviations calculated from technical replicates , n = 3 .

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

C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.


Immunohistochemistry of NSMCE2 on mouse testis . inset illustrates accumulation of NSMCE2 on XY . Scale bar , 50 μm .

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

Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.


Immunofluorescence ( IF ) of NSMCE2 SCP3 on isolated mouse spermatocytes . Threads of NSMCE2 can be detected on regions of weak SCP3 signal , indicative of asynapsis ( arrowheads ) . Scale bar , 2 . 5 μm .

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Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.


Colocalization of NSMCE2 BRCA1 at XY of mouse spermatocytes .

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

Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.


Representative image of NSMCE2 BRCA1 foci in control MMS ‐ treated MEFs . Scale bar , 2 . 5 μm .

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

Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.


of NSMCE2 BRCA1 foci in wild ‐ type MEFs exposed to IR ( 10 Gy ; 1 ‐ h recovery ) , MMS ( 1 mM for 1 h ; 24 ‐ h recovery ) , PARP inhibitor ( 1 μM for 1 h ; 24 ‐ h recovery ) . Data indicate mean values are representative of three independent experiments . Error bars indicate SD .

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

Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.


Representative image of IR ‐ ( 10 Gy ; 1 ‐ h recovery ) MMS ‐ ( 1 mM for 1 ‐ h ; 24 ‐ h recovery ) induced NSMCE2 γH2AX foci in MEFs . Scale bar , 2 . 5 μm .

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Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.


Brightfield microscopy of 2 . 5 dpc obtained from crosses of Nsmce2GT / + mice . right panel is of same 2 shown on left panel , 36 h after being cultured in vitro . arrow indicates that showed asymmetric sizes when isolated , which degenerated upon culture . These were identified as Nsmce2GT / GT through genotyping .

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Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.


Examples of three genotypes found at Mendelian frequencies ( see Appendix Table S1 ) on 2 . 5 dpc .

Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.


Examples of type of nuclei found on Nsmce2GT / GT Nsmce2 + ( + / + or + / GT ) 2 . 5 dpc , 12 h after in vitro culture . Mutant were identified due to absence of NSMCE2 signal ( green ) . DAPI was used to stain ( blue ) . arrows indicate examples of with condensed chromosomes or irregular shapes , which are hardly seen in wild type .

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Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.


Immunoprecipitation of SMC5 ( IgG , as a control ) from a 48 ‐ h culture of B lymphocytes of wt Nsmce2SD / SD . NSMCE2 SMC6 on immunoprecipitates were analyzed by WB . image of Ponceau is shown as a loading control .

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

Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.


IF of NSMCE2 foci in MMS ‐ treated Nsmce2 + / + Nsmce2SD / SD MEFs . Scale bar , 2 . 5 μm .

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IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.


Representative picture of 6 ‐ month ‐ old Nsmce2 + / + Nsmce2SD / SD .

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Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.


Kaplan - Meyer survival curves of Nsmce2 + / + Nsmce2SD / SD mice . P ‐ value was calculated with Mantel - Cox long ‐ rank .

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Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.


A Kaplan - Meyer survival curves of Nsmce2 + / + Nsmce2 + / GT mice . P ‐ value was calculated with Mantel - Cox long ‐ rank . * * * P 0 . 001 .

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A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001.


B incidence found on Nsmce2 + / + Nsmce2 + / GT mice at time of ( i . e . moribund mice ) .

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

B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).


C NSMCE2 evaluated by Western blot in 2 independent pairs of Nsmce2 + / + Nsmce2 + / GT MEFs . β ‐ actin was used as a loading control . Numbers shown bellow indicate relative amounts of NSMCE2 in each sample .

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

C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.


D Sister chromatid exchange ( SCE ) events found per metaphase on Nsmce2 + / + Nsmce2 + / GT MEFs . * * * P 0 . 001 .

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D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.


E , F Relative percentages of micronuclei ( E ) polynucleated ( F ) on cultures of Nsmce2 + / + Nsmce2 + / GT MEFs . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


NSMCE2 assessed by WB in UQ . CreERT2 transgenic Nsmce2 + / + Nsmce2lox / lox littermate MEFs , 24 h after exposure to 4 ‐ OHT . Tubulin are shown as a loading control .

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

NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.


Clonogenic on immortalized UQ . CreERT2 / Nsmce2lox / lox MEFs . picture shows colonies detected after 10 days of growth in presence or absence of 4 ‐ OHT , upon with crystal violet .

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Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.


NSMCE2 BRCA1 foci in MMS ‐ treated ( 1 mM for 1 h ; 24 ‐ h recovery ) UQ . CreERT2 / Nsmce2lox / lox MEFs , 48 h after being treated ( or not ) with 4 ‐ OHT . Scale bar , 5 μm .

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

NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.


SCE events per metaphase on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in presence of 4 ‐ OHT for 72 h . * * * P 0 . 001 .

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

SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.


Example of SCE events ( arrows ) found on Nsmce2 ‐ deleted MEFs .

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Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.


SCE events at telomeres on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in presence of 4 ‐ OHT for 72 h , as detected by CO ‐ FISH ( see ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


Telomere lengths of UQ . CreERT2 Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h .

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

Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.


Example of of telomere signals ( yellow ) used for ( H ) .

Example of the intensity of telomere signals (yellow) used for (H).


of with micronuclei found on cultures of UQ . CreERT2 / Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Intrauterine dwarfism observed upon NSMCE2 deletion on 17 . 5 dpc . Pregnant were treated at 14 . 5 dpc for 3 days with intraperitoneal injections of 4 ‐ OHT . image represents difference in observed between Nsmce2 + / + Nsmce2lox / lox carrying UQ . CreERT2 transgene ( Nsmce2 + / + Nsmce2lox / lox , from now on ) .

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Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).


Representative picture of Nsmce2 + / + Nsmce2lox / lox fed for 40 with a 4 ‐ OHT ‐ containing diet ( see also Movies EV1 EV2 ) .

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Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).


illustrating presence of altered pigmentation problems that arise on Nsmce2lox / lox mice .

Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.


of fat against total mass on Nsmce2 + / + Nsmce2lox / lox . * * * P 0 . 001 .

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Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


bloodcell ( WBC ) from Nsmce2 + / + Nsmce2lox / lox . * * * P 0 . 001 .

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

White bloodcell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


of γH2AX ‐ positive micronuclei found on intestinal epithelia of Nsmce2 + / + Nsmce2lox / lox ( see Appendix Fig S11 ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


A WB illustrating depletion of NSMCE2 in B isolated from 2 ‐ month ‐ old CD19 + / Cre of indicated genotypes , 48 h after being stimulated in vitro with lipopolysaccharide ( LPS ) . Tubulin was used as a loading control .

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A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.


B Representative FACS from B ‐ cultures mentioned in ( A ) . content was measured with propidium iodide ( PI ) . Note presence of with > 4n content in NSMCE2 ‐ deleted .

O O O O O O O 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

B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.


C , D Frequency of origin firing ( C ) fork rate ( D ) were analyzed by fiber analyses ( see ) from prepared from B ‐ cultures explained in ( A ) . No significant differences were observed between Nsmce2 + / + Nsmce2lox / loxB . Data are representative of two independent experiments . Error bars indicate SD . Between 200 300 tracks were measured to estimate fork rate 300 - 500 for calculation of origin firing frequencies .

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C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies.


E Representative of used for explained in ( C , D ) . CldU ( red ) IdU ( green ) channels are shown .

O 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 Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.


Representative pictures of spleens from 2 ‐ month ‐ old CD19 + / Cre of indicated genotypes .

Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.


Numbers of B T from 2 ‐ month ‐ old CD19 + / Cre of indicated genotypes evaluated by flow cytometry ( B220 : B ‐ marker ; CD3 : T ‐ marker ) .

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 B-MEASURED_VAR O O O O O O

Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).


SCE events per metaphase on B obtained from CD19 + / Cre of indicated genotypes , 72 h after being stimulated in vitro with LPS . * * P 0 . 01 , * * * P 0 . 001 .

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SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.


of forward ( FSC ) side ( SSC ) scatter parameters of B obtained from CD19 + / Cre of indicated genotypes , 48 h after being stimulated in vitro with LPS . emergence of bigger is noticeable on lacking both BLM NSMCE2 .

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

Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.


Representative of B ‐ cultures of indicated genotypes 48 h after being stimulated in vitro with LPS . Insets provide a magnified view of an ( arrowhead ) for better comparison of observed differences in . Scale bar , 100 μm .

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

Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.


Examples of types of nuclei found on Blm Nsmce2 double ‐ mutant B ( compared with wild ‐ type B ) , in obtained from ( E ) . DAPI was used to stain . Whereas wt nuclei are regular in , double ‐ mutant invariably presented enlarged , multilobulated , irregular nuclei . Scale bar , 2 . 5 μm .

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

Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.


B Western blot analyses after induced expression of FLAG - tagged UPF3A in WT HEK293 HeLa ( n = 1 ) . Tubulin serves as control . asterisk indicates unspecific bands .

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

B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands.


C Fraction of expressed genes ( genes with non - zero in DESeq2 ) were calculated which exhibit or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) / or alternative splicing ( AS ) events in HEK293 HeLa WT overexpressing UPF3A using respective computational ( cutoffs are indicated ) . AS DTU events were collapsed on gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald corrected for multiple testing using Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR ( ISAR ) using a DEXSeq - corrected for multiple testing using Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution corrected for multiple testing using Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D Volcano plot showing differential gene expression analyses from - Seq dataset of HEK293 HeLa WT overexpressing UPF3A . log2 fold change is plotted against - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald corrected for multiple testing using Benjamini - Hochberg method . OE = overexpression .

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

D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.


E Read coverage of SMG5 from WT HEK293 HeLa - seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer ( IGV ) snapshot . Differential gene expression ( from DESeq2 ) is indicated as log2 fold change ( log2FC ) on right . Schematic representation of coding transcript below .

O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.


A Western blot of WT UPF3A KO ( clones 4 , 14 20 ) with indicated siRNA treatments ( n = 1 ) . UPF3A UPF3B were detected , Tubulin serves as control . asterisk indicates unspecific bands .

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

A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of indicated lines treated with indicated siRNAs for 2 or 6 days . For RSRC2 SRSF2 of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points means are plotted as log2 fold change ( log2FC ) ( n = 3 for RSRC2 SRSF2 , n = 4 for ZFAS1 ) .

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

B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).


C Fraction of expressed genes ( genes with non - zero in DESeq2 ) were calculated which exhibit or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) / or alternative splicing ( AS ) events in indicated conditions using respective computational ( cutoffs are indicated ) . AS DTU events were collapsed on gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald corrected for multiple testing using Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - corrected for multiple testing using Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution corrected for multiple testing using Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D , E Volcano plots showing differential gene expression analyses from indicated - Seq datasets ( D UPF3A KO clone 14 , E UPF3A KO clone 20 ) . log2 fold change is plotted against - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald corrected for multiple testing using Benjamini - Hochberg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method


A Western blot of WT UPF3B KO ( clones 90 91 ) combined with indicated knockdowns ( n = 1 ) . UPF3A UPF3B ( AK - 141 ) were detected , Tubulin serves as control . of UPF3A were quantified , normalized to tubulin expression , shown as datapoints mean . Fold - changes of relevant conditions are shown . asterisk indicates unspecific bands .

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

A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of indicated lines with indicated knockdowns . For RSRC2 SRSF2 of NMD isoform to canonical isoform was calculated . Data points means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3).


D , E Volcano plots showing differential transcript usage ( via IsoformSwitchAnalyzeR ) in various - Seq data . Isoforms containing GENCODE ( release 33 ) annotated PTC ( red , TRUE ) , regular stop codons ( blue , FALSE ) or having no annotated open reading frame ( grey , NA ) are indicated . change in isoform fraction ( dIF ) is plotted against - log10 adjusted p - value ( adj . p - value ) . Density plots show distribution of filtered isoforms in respect to dIF , cutoffs were | dIF | > 0 . 1 adj . p - value < 0 . 05 . P - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - corrected for multiple testing using Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.


A Western blot of WT , UPF3B KO UPF3A - UPF3B double KO ( clones 1 2 ) ( n = 1 ) . UPF3A UPF3B were detected , Tubulin serves as control . asterisk indicates unspecific bands .

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

A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


C Quantitative RT - PCR of indicated samples with indicated KDs . For RSRC2 SRSF2 of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

C Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


E Northern blot of globin reporter xrFrag . Ethidium bromide stained 28S 18S rRNAs are shown as controls . results are shown as data points mean ( n = 3 ) .

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

E Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).


A - C Volcano plots of label free mass spectrometry - of interaction partners of UPF2 in WT treated with control siRNAs UPF3B KO clone 90 dKO clone 1 both treated with siRNAs targeting UPF3B ( n = 4 biologically independent samples ) . ( A ) FLAG - UPF2 in WT against FLAG - GST control in WT , ( B ) UPF2 in 3B KO against FLAG control in WT , ( C ) UPF2 in dKO against FLAG control in WT . Points labeled in purple indicate NMD factors ; points labeled in turquoise indicate EJC components . Cut offs : Log2 fold change ≥

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

A-C Volcano plots of label free mass spectrometry-based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B (n = 4 biologically independent samples). (A) FLAG-UPF2 in WT against FLAG-GST control in WT cells, (B) UPF2 in 3B KO cells against FLAG control in WT cells, (C) UPF2 in dKO cells against FLAG control in WT cells. Points labeled in purple indicate NMD factors; points labeled in turquoise indicate EJC components. Cut offs: Log2 fold change ≥ 0


E Representative Western blot after FLAG co - immunoprecipitation ( IP ) of FLAG - tagged GST ( control ) or UPF3B WT single mutant constructs in UPF3B KO ( n = 3 ) .

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

E Representative Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or UPF3B WT and single mutant constructs in UPF3B KO cells (n = 3).


F Western blot of WT UPF3B KO clone 90 with Luciferase UPF3A KDs respectively . Monitored expression of FLAG - tagged UPF3B rescue construct shown in ( D ) . Rescue construct were detected with anti - FLAG anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) .

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

F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3B rescue construct shown in (D). Rescue construct protein levels were detected with anti-FLAG and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1).


G Quantitative RT - PCR of samples from ( F ) . For RSRC2 SRSF2 of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points means are plotted as log2 fold change ( log2FC , n = 3 ) .

G Quantitative RT-PCR of the samples from (F). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


B Western blot of WT UPF3B KO clone 90 with Luciferase UPF3A KDs respectively . Monitored expression of FLAG - tagged UPF3A UPF3B rescue construct shown in ( A ) . Rescue construct were detected with anti - FLAG , anti - UPF3A anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) . asterisk indicates unspecific bands .

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

B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3A and UPF3B rescue construct shown in (A). Rescue construct protein levels were detected with anti-FLAG, anti-UPF3A and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1). The asterisk indicates unspecific bands.


C Quantitative RT - PCR of samples from ( B ) . For RSRC2 SRSF2 of NMD isoform to canonical isoform was calculated . Data points means are plotted as log2 fold change ( log2FC ) ( n = 3 ) .

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

C Quantitative RT-PCR of the samples from (B). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC) (n=3).


B Representative adhesion frequency ( Pa ) vs . contact duration ( tc ) curves forNKG2D expressing NK ( n ≥ 3 ) in contact with RBCs ( n ≥ 3 ) coated with a ligand ( MICA in red , MICB in orange , ULBP1 in green , or ULBP3 in blue ) at different contact durations , fitted by non - linear in - situ binding kinetics model Site densities of NKG2D ( mr ) its ligands ( ml ) are indicated . Data information : Error bars are mean ± for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - unpaired 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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

B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free affinities ( C ) , on - rates ( D ) of NKG2D binding with indicated ligands from mammalian . in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - unpaired t - ) .

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

In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free off - rates ( E ) of NKG2D binding with indicated ligands from mammalian . in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - unpaired t - ) .

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

In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


F Detection range comparison in affinity measurement of NKG2D indicated ligands between in - situ in - solution . Bars in different colors are ratios of affinities of indicated ligands divided by that of ULBP1 . Data information : In - solution affinities of purified from E . coli In - solution affinities of purified from mammalian were measured by

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

F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI


IFN - γ release IFN - γ production ( B ) by human peripheral NK stimulated with plate - coated NKG2D ligands ( MICA in red , MICB in green ULBP3 in blue ) assessed by Cytometric Bead Array , of which are their 50 % effective ( EC50 ) ( C ) . Data information : IFN - γ release in ( B ) was one representative experiment of three total independent experiments . Data points in ( B ) bars in ( C ) represent mean values . Error bars in ( B ) ( C ) represent mean ± . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - unpaired t - ) .

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

IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


D - F Plots of reciprocals of EC50 vs . effective in - situ affinities ( AcKa ) ( D ) , effective in - situ on - rate ( AcKon ) ( E ) in - situ off - rate ( koff ) ( 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

D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).


C Verification of binding specificity of NKG2D with different ligands . Data information : Every dot represents adhesion frequency of one - bead pair experimental data came from at least two to three repeated trials . Horizontal lines represent mean values . Error bars represent mean ± .

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

C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM.


E , F Force - dependent bond lifetimes of NKG2D various ligands at 5 pN , 10 pN 15 pN ( E ) full force spectrum ( F ) . Data information : Bond lifetimes in ( E ) ( F ) ( in which n = 1505 for MICA , n = 1852 for MICB , n = 531 for ULBP1 , n = 1241 for ULBP3 ) of ligands with NKG2D came from at least 19 NK - bead pairs of at least four independent biological experiments . bars in ( E ) , data points in ( F ) represent mean values . Error bars represent mean ± .

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

E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM.


C force vs . extension trajectories for simulations are shown in ( A ) ( B ) . Occurrence of sudden extension changes are indicated in shaded time points correspond with snapshots in ( A ) ( B ) are marked .

O O O O O O O O O O O O O O O O O O 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 The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.


F vs . time curves for force - induced H - bond formation between indicated residues within NKG2D - MICA binding interfaces . dashed red lines represent H - bonds whose distances are less than 3 . 5 Å .

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

F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.


A , B Abolishments of force - induced binding residues by 3A mutations ( MICA 3A , n = 526 ) TAT mutations ( MICA TAT , n = 580 ) impairs NKG2D ' s catch bond with MICA WT ( n = 1505 ) ( A ) IFN - γ release ( B ) . Data information : Bond lifetimes in ( A ) of MICA WT mutants with NKG2D came from at least 21 pairs of beads of at least three repeated experiments . IFN - γ release of NK in ( B ) was one representative experiment of three total independent experiments . Data points represent mean values . Error bars represent mean ± for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - unpaired t - ) .

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

A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


50 % effective ( EC50 ) of NK stimulated by indicated MICA mutants Data information : horizontal lines represent mean values . Error bars represent mean ± for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - unpaired t - ) .

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

50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


IFN - γ release of NK stimulated by indicated MICA mutants at a of 11nM ( D ) 22nM ( E ) . Data information : bars in ( E ) , data point in ( F ) represent mean values . Error bars represent mean ± for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - unpaired t - ) .

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

IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


A - C Plots Pearson correlation of reciprocals of EC50 vs . force - dependent in - situ koff at 5 pN ( A ) , 10 pN ( B ) 15pN ( C ) .

O O O O O O O O O O O O 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-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).


I Detection range comparison in force - dependent affinity of NKG2D indicated ligands under difference force . Bars in different colors are ratios of affinities of indicated ligands divided by that of ULBP1 .

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 B-MEASURED_VAR O

I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.


D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on - rates force - dependent off - rates .

O 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 Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on-rates and force-dependent off-rates.


( A ) Assessment of vacuole ( arrows ) formation in neutrophils after stimulation with PMA ( II ) rapamycin ( III ) , compared with control ( I : treated with PBS serum ) ( magnification 1000 × ) .

O O O O O O O O O O O O O O O B-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

(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).


( B ) ( I ) Presence of late autophagosomes as observed with MDC in treated with PMA rapamycin ( RAP ) compared with control ( PBS ) treated with PMA 3 ‐ MA ( magnification 400 1000 × ) . ( II ) of PMN with acidified vacuoles as detected by MDC . Data are representative of six independent experiments are presented as mean ± SD ; Wilcoxon matched ‐ pairs , * p < 0 . 05 : PMA ( bar 2 : 86 . 16 ± 6 . 49 % ) , rapamycin ( bar 3 : 65 . 83 ± 8 . 47 % ) treated , treated with PMA 3 ‐ MA ( bar 4 : 9 . 17 ± 0 . 75 % ) treated with rapamycin 3 ‐ MA ( bar 5 : 8 . 5 ± 1 . 87 % ) compared with control conditions ( bar 1 : 15 . 67 ± 3 . 50 % ) . of with acidified autophagosomes in PMN treated with PMA 3 ‐ MA ( bar 4 ) or rapamycin 3 ‐ MA ( bar 5 ) was significantly lower ( p < 0 . 05 ) compared with treated with PMA ( bar 2 ) rapamycin ( bar 3 ) , respectively .

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

(B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.


( C ) of LC3B ‐ I to LC3B ‐ II conversion in treated with PMA ( lane IV ) rapamycin ( lane II ) , as observed by LC3B immunoblotting . Effect of 3 ‐ MA in both PMA ( lane V ) rapamycin ( lane I ) ‐ treated . Lane III : control , lane VI : treated only with 3 ‐ MA . One representative out of six independent experiments is shown .

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

(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.


( D ) Impact of PI ‐ 3K inhibition using LY294002 on LC3B conversion after PMA challenge ( lane III ) , ( lane I : control , lane II : PMA , lane IV : LY294002 only ) . One representative out of four independent experiments is shown .

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

(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.


( E ) Immunofluorescence confocal microscopy of LC3B . ( I ) Control treated with PBS serum , ( II ) PMA ‐ treated . 1 . DAPI : blue , 2 . LC3B : red 3 . merge . ( III ) of PMN with LC3B . Control conditions ( bar 1 : 17 . 7 ± 2 . 4 % ) , 3 ‐ MA ( bar 2 : 3 ± 1 . 01 % ) , PMA ( bar 3 : 69 ± 2 . 1 % ) , PMA 3 ‐ MA ( bar 4 : 4 ± 1 . 9 % ) , rapamycin ( bar 5 : 56 ± 1 . 6 % ) . Data are representative of five independent experiments presented as mean ± SD ; Student ' s t ‐ , * p < 0 . 01 compared to control ( bar 1 ) or indicated comparision ( bar 3 vs 4 ) .

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

(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).


ATG3 LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin . ATG3 mRNA expression ( fold ) after PMA ( 2 . 44 ± 0 . 78 ) rapamycin ( RAP : 1 . 81 ± 0 . 41 ) stimulation . LC3B gene alterations ( PMA : 4 . 98 ± 0 . 59 ) rapamycin ( RAP : 1 . 7 ± 0 . 55 ) . Effect of 3 ‐ MA pre ‐ treatment , for both stimuli , was also assessed ( PMA + 3 ‐ MA : ATG3 : 1 . 36 ± 0 . 64 , LC3B : 1 . 5 ± 0 . 49 rapamycin + 3 ‐ MA : ATG3 : 1 . 28 ± 0 . 46 , LC3B : 1 . 24 ± 0 . 57 ) . No other significant alteration in ATG genes mRNA was detected . Representative data of ATG7 mRNA expression after treatment with PMA ( 1 . 21 ± 0 . 42 ) rapamycin ( 0 . 94 ± 0 . 64 ) . Data are representative of six independent experiments presented as mean relative expression ± SD . Wilcoxon matched ‐ pairs , * p < 0 . 05 n . s . = not significant compared with control conditions ( dot ) as indicated in figure . Relative expression ( folds ) of each gene is derived from substitution of DCT values ( normalized by GAPDH DCT value ) in 2DDCT equation 36 .

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

ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.


( A ) ( I ) MDC in neutrophils treated with LPS , LTA , peptidoglycan ( PGN ) , loxorubin ( LOX ) R848 ( 400 × ) . ( II ) of PMN with acidified vacuoles as detected by MDC . LPS ( 50 ng / mL ) ( bar 2 : 69 . 67 ± 8 . 21 % ) , LTA ( bar 3 : 75 . 17 ± 9 . 80 % ) , peptidoglycan ( bar 4 : 77 ± 6 . 96 % ) , loxorubin ( bar 5 : 66 . 67 ± 5 . 61 % ) , R848 ( bar 6 : 69 . 5 ± 7 . 45 % ) , poly I : C ( bar 7 : 16 . 33 ± 3 . 14 % ) flagellin ( bar 8 : 19 . 83 ± 5 . 16 % ) − treated compared with control conditions ( bar 1 : 14 . 5 ± 4 . 97 % ) . Data are representative of six independent experiments presented as mean ± SD ; Wilcoxon matched − pairs , * p < 0 . 05 , n . s . = not significant .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.


( B ) LC3B lipidation status of treated with LPS ( lane I ) , LTA ( lane II ) , peptidoglycan ( lane III ) , loxoribin ( lane IV ) R848 ( lane V ) , detected by immunoblotting . One representative out of six independent experiments is shown .

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

(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.


( C ) LC3B − II / LC3B - I as obtained from integrated optical density measurements . LTA ( bar 2 : 1 . 75 ± 0 . 84 ) , peptidoglycan ( bar 3 : 1 . 82 ± 1 . 17 ) , LPS ( bar 4 : 1 . 9 ± 1 . 7 ) , loxorubin ( bar 5 : 1 . 32 ± 0 . 49 ) R848 ( bar 6 : 1 . 4 ± 0 . 77 ) compared with control neutrophils ( bar 1 : 0 . 76 ± 0 . 30 ) . Data are representative of six independent experiments are presented as mean ± SD . Wilcoxon matched − pairs , * p < 0 . 05 .

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

(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.


( D ) of LC3B gene expression in neutrophils treated with TLR agonists . LPS ( bar 1 : 2 . 27 ± 0 . 43 ) , peptidoglycan ( bar 2 : 2 . 07 ± 0 . 42 ) , LTA ( bar 3 : 2 . 1 ± 0 . 49 ) R848 ( bar 4 : 1 . 89 ± 0 . 35 ) compared with medium − treated control . Dotted represents LC3B mRNA of medium - treated control . Data are representative of six independent experiments are presented as mean relative expression ± SD . Wilcoxon matched − pairs , * p < 0 . 05 .

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

(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( E ) Assessment of ATG gene expression in PMN after LPS stimulation ( 50 ng / mL ) . ATG3 ( bar 1 : 2 . 94 ± 0 . 52 ) , ATG4 ( bar 2 : 1 . 72 ± 0 . 42 ) , ATG5 ( bar 3 : 1 . 84 ± 0 . 34 ) , ATG6 ( bar 4 : 1 . 8 ± 0 . 3 ) , ATG7 ( bar 5 : 3 . 13 ± 0 . 64 ) LC3B ( bar 6 : 2 . 27 ± 0 . 43 ) compared with control . Dotted represents mRNA of medium - treated . Data are representative of six independent experiments are presented as mean relative expression ± SD . Wilcoxon matched − pairs , * p < 0 . 05 .

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

(E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( A ) ( I ) Formation of acidified autophagosomes after stimulation with 10 ng / mL of IL ‐ 1β , compared with medium ‐ treated control . ( II ) of PMN with acidified vacuoles as detected by MDC . IL ‐ 1β ‐ treated ( bar 2 : 65 . 17 ± 10 . 23 % ) compared with control conditions ( bar 1 : 15 . 83 ± 6 . 08 % ) . Data are representative of six independent experiments presented as mean ± SD ; Wilcoxon matched ‐ pairs , * p < 0 . 05 .

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

(A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


( B ) LC3B conversion in neutrophils treated with 10 ng / mL ( lane II ) 100 ng / mL ( lane III ) of IL ‐ 1β . ( lane I : medium ‐ treated control ) . One representative out of five independent experiments is shown .

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

(B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.