Representative images of human respiratory tissues , all stained on consecutive sections , with immunohistochemistry using two antibodies targeting human ACE2 protein ( brown ) , and counterstained with hematoxylin ( blue ) . Respiratory tissues were composed of different structures in nasal mucosa , bronchus , smaller bronchioles and lung tissue . ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium ( arrowheads ) . Rare ACE2 staining was present in a few alveolar cells ( arrows ) . No staining was observed in nasal squamous epithelium , bronchioles , or submucosal glands in either tissue . Gender and age are shown for all individuals . Red and green colored squares mark the positions in the TMA cores shown as magnifications . Scale bar = 50µm . Scale bar for images in dashed squares = 10 µm . ( re = respiratory ; sq = squamous ) .

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Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).


( B ) ACE2 protein abundance in different human tissues from ProteomicsDB , with the median expression presented in log10 ( iBAQ ) .

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(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).


( C ) Western blot of ACE2 using five different human tissue lysates . For lung , two different lysates were used : one male ( left ) and one female ( right ) .

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(C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).


H & E stained sections of back skin from indicated mice treated with IMQ for 2 , 3 or 5 days . Bright - field images , Magnification : 20x , Scale bar : 100 µm .

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H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3 or 5 days. Bright-field images, Magnification: 20x, Scale bar: 100 µm.


Flow cytometry of total back skin after 2 , 3 and 5 days of IMQ treatment . Analyzed were dermal γδ T cells ( γδ TCRint + ) ( F ) Graphs show immune cells as % of live , single cells . ( n = 10 - 28 ( F ) , ; ≥ 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by Bonferroni multiple comparison test Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . See Appendix Table S3 for exact P ‐ values .

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Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+) (F) Graphs show immune cells as % of live, single cells. (n = 10-28 (F), ; ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.


Flow cytometry of total back skin after 2 , 3 and 5 days of IMQ treatment . Analyzed were Monocytes ( CD11b + Ly6Chi ) ( G ) , and Neutrophils ( CD11b + Ly6G + ) ( H ) . Graphs show immune cells as % of live , single cells . n = 10 - 21 ( G ) , n = 4 - 25 ( H ) ; ≥ 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by Bonferroni multiple comparison test Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . See Appendix Table S3 for exact P ‐ values .

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Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were Monocytes (CD11b+Ly6Chi) (G), and Neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells. n = 10-21 (G), n = 4-25 (H); ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.


H - I . Western blot analysis of Phospho - or total JNK and c - Jun in BMDCs of indicated genotype pre - treated with DMSO ( 1 : 1000 ) or SP600125 ( JNKi , 25 µM ) for 1h and stimulated with IMQ ( 5 µg / mL ) for 15 min .

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H-I. Western blot analysis of Phospho- or total JNK and c-Jun in BMDCs of indicated genotype pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with IMQ (5 µg/mL) for 15 min.


Flow cytometry of back skin 12h after intradermal ( i . d . ) injection of 1 µg rCCL2 and / or IMQ treatment . Analyzed were pDCs ( BST - 2 + B220 + CD11cintCD11b - ) shown as % of live , single cells . ( n = 6 ; 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by Tukey multiple comparison test . Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . See Appendix Table S3 for exact P ‐ values .

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

Flow cytometry of back skin 12h after intradermal (i.d.) injection of 1 µg rCCL2 and/or IMQ treatment. Analyzed were pDCs (BST-2+B220+CD11cintCD11b-) shown as % of live, single cells. (n = 6; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Tukey multiple comparison test. Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.


Luciferase activity was quantified in lysates of RAW 264 . 7 cells transfected with pGL3 basic vector harboring wild - type ( wt ) or mutated ( mut1 or 2 ) IL - 23p19 promoter . Transfected cells were pre - treated with JNKi ( SP600125 , 25 µM , 1h ) and stimulated with IMQ ( 6h ) . Results are shown as fold change to pGL - 3 - wt transfected , LAL ( Limulus amebocyte lysate ) treated RAW264 . 7 cells . ( n = 4 ; 2 independent experiments ) .

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

Luciferase activity was quantified in lysates of RAW 264.7 cells transfected with pGL3 basic vector harboring wild-type (wt) or mutated (mut1 or 2) IL-23p19 promoter. Transfected cells were pre-treated with JNKi (SP600125, 25 µM, 1h) and stimulated with IMQ (6h). Results are shown as fold change to pGL-3-wt transfected, LAL (Limulus amebocyte lysate) treated RAW264.7 cells. (n = 4; 2 independent experiments).


Epidermal thickness of back skin was quantified in H & E stained sections of indicated mice treated with rIL - 23 ( i . d . , 1µg ) and / or IMQ for two consecutive days . ( n = 4 - 7 ; 3 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by one - way ANOVA with Tukey multiple comparison test Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . See Appendix Table S3 for exact P ‐ values .

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Epidermal thickness of back skin was quantified in H&E stained sections of indicated mice treated with rIL-23 (i.d., 1µg) and/or IMQ for two consecutive days. (n = 4-7; 3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.


DC subsets ( cDC1 , cDC2 ) , Macrophages ( MP ) and Langerhans cells ( LC ) were sorted from IMQ treated back skin ( 5h ) . c - Jun , Ccl2 and Il23p19 mRNA was analyzed by qRT - PCR . Sort strategy is shown in ( B ) . ( 10 mice were pooled per condition , 1 experiment ) .

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

DC subsets (cDC1, cDC2), Macrophages (MP) and Langerhans cells (LC) were sorted from IMQ treated back skin (5h). c-Jun, Ccl2 and Il23p19 mRNA was analyzed by qRT-PCR. Sort strategy is shown in (B). (10 mice were pooled per condition, 1 experiment).


Dermal γδ T cells ( γδ TCRint + ) , Monocytes ( CD11b + Ly6Chi ) , and Neutrophils ( CD11b + Ly6G + ) were analyzed by flow cytometry in mouse ear at the end of treatment ( Day 28 ) . Graphs show immune cells as % of live , single cells . ( n = 2 - 8 , 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by Bonferroni multiple comparison test ( N ) . Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . See Appendix Table S3 for exact P ‐ values .

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Dermal γδ T cells (γδ TCRint+), Monocytes (CD11b+Ly6Chi), and Neutrophils (CD11b+Ly6G+) were analyzed by flow cytometry in mouse ear at the end of treatment (Day 28). Graphs show immune cells as % of live, single cells. (n = 2-8, 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test (N). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.


Immunofluorescence of c - Jun ( green ) , CD1a or CD1c or CD14 ( red ) and DAPI in psoriatic lesions . Shown is a representative image ( Magnification 25x , Scale Bar : 100µm ) with an Inset ( Magnification 63x , Scale Bar : 20 µm , Deconvoluted ) . Arrows indicate c - Jun + DCs . Asterisk highlights a DC with prominent c - Jun expression as shown enlarged in 2 white - framed windows ( DC Marker ( red ) + c - Jun ( green , left ) or DAPI ( blue , right ) ) .

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

Immunofluorescence of c-Jun (green), CD1a or CD1c or CD14 (red) and DAPI in psoriatic lesions. Shown is a representative image (Magnification 25x, Scale Bar: 100µm) with an Inset (Magnification 63x, Scale Bar: 20 µm, Deconvoluted). Arrows indicate c-Jun+ DCs. Asterisk highlights a DC with prominent c-Jun expression as shown enlarged in 2 white-framed windows (DC Marker (red) + c-Jun (green, left) or DAPI (blue, right)).


D - E . Representative immunofluorescence of c - Jun ( green ) , CD1a ( red ) , CCL2 ( D ) or Il23p19 ( E ) ( white ) and DAPI in psoriatic lesions . Arrows indicate triple - positive cells and an asterisk highlights a representative one that is shown enlarged in a white - framed inlet . Magnification : 40x . Scale bar : 50 µm . ( n = 2 patient samples ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).


B - C Human mo - DCs pre - treated with JNKi ( SP600125 , 25µM ) ( B ) or AP - 1 Inhibitor ( T - 5224 , 20 µM ) ( C ) for 1h were stimulated with R848 ( 10 µg / ml ) . CCL2 and IL - 23 were analyzed by ELISA . ( n = 5 - 11 ; 2 - 3 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by paired , two - tailed t - test ( B , C , Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.


E - F Human moDCs described in ( D ) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry ( E ) and CCL2 or IL - 23 protein by ELISA ( F ) . ( n = 7 ; 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by paired , two - tailed t - test E and F ) . Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.


Immunoblot of media and lysate collected from HEK293T cells overexpressing ERdj3WT or ERdj3KDEL . Fresh media was conditioned on cells for 24 h prior to harvest .

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Immunoblot of media and lysate collected from HEK293T cells overexpressing ERdj3WT or ERdj3KDEL. Fresh media was conditioned on cells for 24 h prior to harvest.


qPCR analysis of ERdj3 mRNA in HEK293T cells treated with or without thapsigargin ( Tg ; 6 h , 100 nM ) . Error bars represent the mean ± 95 % confidence interval as calculated in DataAssist 2 . 0 ( n = 3 ) .

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qPCR analysis of ERdj3 mRNA in HEK293T cells treated with or without thapsigargin (Tg; 6 h, 100 nM). Error bars represent the mean ± 95% confidence interval as calculated in DataAssist 2.0 (n = 3).


Representative immunoblot of lysates and media collected from HEK293T - Rex cells stably expressing non - silencing ( NS ) or ERdj3 shRNA . Cells were treated with the indicated concentration of Tg in fresh media for 16 h . Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T - Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C . * indicates a pvalue < 0 . 05 ; ( n = 3 ) .

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

Representative immunoblot of lysates and media collected from HEK293T-Rex cells stably expressing non-silencing (NS) or ERdj3 shRNA. Cells were treated with the indicated concentration of Tg in fresh media for 16 h. Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T-Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C. *indicates a pvalue < 0.05; (n = 3).


Representative autoradiogram of [ 35S ] - labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg ( 1 µM ) . The experimental paradigm for labeling is shown above . Quantification of fraction intracellular ( dark grey ) and extracellular ( light grey ) [ 35S ] - labeled ERdj3 following a 4 h chase relative to total [ 35S ] - labeled ERdj3 at 4 h ( as shown in Figure 1E ) . Error bars represent standard error from biological replicates ( n = 3 ) . Quantification of total [ 35S ] - labeled ERdj3 in media collected from cells pretreated with vehicle or Tg , as shown in Figure 1E . The media [ 35S ] - labeled ERdj3 was measured by densitometry and is normalized to the amount of media [ 35S ] - labeled ERdj3 at 4 h from vehicle - treated cells . Error bars represent standard error from biological replicates . * indicates a pvalue < 0 . 05 ; ( n = 3 ) .

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

Representative autoradiogram of [35S]-labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg (1 µM). The experimental paradigm for labeling is shown above. Quantification of fraction intracellular (dark grey) and extracellular (light grey) [35S]-labeled ERdj3 following a 4 h chase relative to total [35S]-labeled ERdj3 at 4 h (as shown in Figure 1E). Error bars represent standard error from biological replicates (n = 3). Quantification of total [35S]-labeled ERdj3 in media collected from cells pretreated with vehicle or Tg, as shown in Figure 1E. The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 4 h from vehicle-treated cells. Error bars represent standard error from biological replicates. *indicates a pvalue < 0.05; (n = 3).


Representative immunoblot and quantification of sera collected at the indicated times from DBA / 2J mice fed a high fat ( 60 % fat ) diet after a 18 h fast . A schematic of the fasting / refeeding experimental setup is shown above . The quantification was normalized for each mouse to the amount of ERdj3 in the serum draw immediately prior the fast . * indicates a p - value < 0 . 05 ; ( n = 6 ) .

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Representative immunoblot and quantification of sera collected at the indicated times from DBA/2J mice fed a high fat (60% fat) diet after a 18 h fast. A schematic of the fasting/refeeding experimental setup is shown above. The quantification was normalized for each mouse to the amount of ERdj3 in the serum draw immediately prior the fast. *indicates a p-value < 0.05; (n = 6).


Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [ by 1 g / mL dox ] and / or ATF6 [ by 10 M TMP ] , as indicated ( Shoulders et al , 2013 ) . Immunoblots of the ATF6 target protein BiP ( reactive with the KDEL antibody ) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors .

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Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.


Representative autoradiogram of [ 35S ] - labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [ by 1 g / mL dox ] and / or ATF6 [ by 10 M TMP ] ( Shoulders et al , 2013 ) . The experimental protocol is shown above . Quantification of relative amounts of [ 35S ] - labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B ( n = 3 ) . The media [ 35S ] - labeled ERdj3 was measured by densitometry and is normalized to the amount of media [ 35S ] - labeled ERdj3 at 8 h from vehicle - treated cells . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 ; * * * indicates p - value < 0 . 001 as compared to the vehicle - treated condition . Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B . The fraction of ERdj3 secreted was calculated by normalizing the recovered [ 35S ] - labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [ 35S ] - labeled ERdj3 signal in the media and lysates at t = 0 h . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 as compared to the vehicle - treated condition . Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B . The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [ 35S ] - labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [ 35S ] - labeled ERdj3 signal in the media and lysates at t = 0 h . Error bars represent standard error from biological replicates ( n = 3 ) . * indicates p - value < 0 . 05 ; * * indicates p - value < 0 . 01 as compared to the vehicle - treated condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 autoradiogram of [35S]-labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP] (Shoulders et al, 2013). The experimental protocol is shown above. Quantification of relative amounts of [35S]-labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B (n=3). The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 8 h from vehicle-treated cells. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01; *** indicates p-value < 0.001 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B. The fraction of ERdj3 secreted was calculated by normalizing the recovered [35S]-labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B. The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [35S]-labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.


Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native ( N ) or guanidinium hydrochloride ( GdnHCl ) - denatured ( D ) RNAse A covalently coupled to sepharose resin . Resin was incubated with media for 2 h at ambient temperature , followed by washing and elution in Laemmli ' s reducing buffer .

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

Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNAse A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and elution in Laemmli's reducing buffer.


Plot showing the time - dependent increase in ThioT fluorescence ( ex : 440 nm , em : 485 nm ) of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation in the presence or absence ( black ) of recombinant ERdj3 ( RERdj3 ) . The RERdj3 concentrations and molar ratios of RERdj3 to A1 - 40 are as follows : Violet : 1 . 5 nM , 1 : 6561 ; light blue : 4 . 6 nM , 1 : 2187 ; dark blue : 14 nM , 1 : 729 ; dark green : 41 nM , 1 : 243 ; light green : 120 nM , 1 : 81 ; yellow : 370 nM , 1 : 27 ; orange : 1 . 1 M , 1 : 9 ; red : 3 . 3 M , 1 : 3 , or maroon : 10 M , 1 : 1 . All traces represent the average of three replicates .

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

Plot showing the time-dependent increase in ThioT fluorescence (ex:440 nm, em:485 nm) of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to A1-40 are as follows: Violet: 1.5 nM, 1:6561; light blue: 4.6 nM, 1:2187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 M, 1:9; red: 3.3 M, 1:3, or maroon: 10 M, 1:1. All traces represent the average of three replicates.


Plot showing the time - dependent increase in ThioT fluorescence of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation in the presence or absence of BSA ( 14 µg / mL ) or RERdj3 ( 14 µg / mL ; 370 nM , RERdj3 : A = 1 : 27 ) . All traces represent the average of three replicates .

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

Plot showing the time-dependent increase in ThioT fluorescence of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence of BSA (14 µg/mL) or RERdj3 (14 µg/mL; 370 nM, RERdj3: A=1:27). All traces represent the average of three replicates.


Immunoblot of A1 - 40 incubated ( 10 µM , 37 OC , pH 7 . 2 ) with regular agitation for 90 h in the presence or absence of RERdj3 ( 14 µg / mL ; 370 nM , RERdj3 : A1 - 40 = 1 : 27 ) or BSA ( 14 µg / mL ) . The reaction solution in the well was collected ( Nonadhered ) and separated into soluble and insoluble fractions by centrifugation . These samples , as well as the washed wells of the plate ( Adhered ) , were denatured in 8 M GdnHCl with sonication and analyzed by SDS - PAGE / immunoblotting .

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

Immunoblot of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation for 90 h in the presence or absence of RERdj3 (14 µg/mL; 370 nM, RERdj3: A1-40 =1:27) or BSA (14 µg/mL). The reaction solution in the well was collected (Nonadhered) and separated into soluble and insoluble fractions by centrifugation. These samples, as well as the washed wells of the plate (Adhered), were denatured in 8 M GdnHCl with sonication and analyzed by SDS-PAGE/immunoblotting.


Representative images under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T - Rex or conditioned media prepared from HEK293T - Rex cells overexpressing ERdj3WT in the presence or absence of TPrP ( 600 ng / mL ) , as described in Figure 4A . Arrows indicate representative cells containing vacuoles . Insets show 544X magnification of a typical region of the image . Quantification of vacuole formation from images as shown in Figure 4B . Equal area images comprising ~ 1200 cells per well were counted for each sample . Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package ( Schindelin et al , 2012 ) . * * * indicates a p - value < 0 . 001 ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T-Rex or conditioned media prepared from HEK293T-Rex cells overexpressing ERdj3WT in the presence or absence of TPrP (600 ng/mL), as described in Figure 4A. Arrows indicate representative cells containing vacuoles. Insets show 544X magnification of a typical region of the image. Quantification of vacuole formation from images as shown in Figure 4B. Equal area images comprising ~1200 cells per well were counted for each sample. Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package (Schindelin et al, 2012). *** indicates a p-value < 0.001 (n = 3).


Immunoblot of M2 anti - FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT , FTTTRA25T , and / or ERdj3WT either as a co - incubation or co - expression experiment as shown in Figure 5A . Beads were washed in RIPA buffer prior to elution . Media inputs ( 1 : 400 ) are shown as a control .

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

Immunoblot of M2 anti-FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT, FTTTRA25T, and/or ERdj3WT either as a co-incubation or co-expression experiment as shown in Figure 5A. Beads were washed in RIPA buffer prior to elution. Media inputs (1:400) are shown as a control.


Immunoblot of M2 anti - FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated . Media inputs ( 1 : 400 ) are shown as a control .

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

Immunoblot of M2 anti-FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated. Media inputs (1:400) are shown as a control.


Immunoblot of M2 anti - FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis , a kinetic stabilizer of TTR tetramers . Media inputs ( 1 : 400 ) are shown as a control .

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

Immunoblot of M2 anti-FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis, a kinetic stabilizer of TTR tetramers. Media inputs (1:400) are shown as a control.


Immunoblot of 6E10 anti - APP immunopurifications from the conditioned media of CHO cells or CHO - derived APP751 - expressing ( 7WD10 ) cells overexpressing ERdj3WT as indicated . Cells were replated together as indicated 24 h after transfection at equal stoichiometry , and media conditioned for 36 h . In this case , ERdj3 transfected CHO cells replated with mock - transfected 7WD10 cells serves as the co - incubation condition , while mock - transfected CHO cells replated with ERdj3 - transfected 7WD10 cells comprise the co - expression condition . Media inputs ( 1 : 150 ) are shown as a control .

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

Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT as indicated. Cells were replated together as indicated 24 h after transfection at equal stoichiometry, and media conditioned for 36 h. In this case, ERdj3 transfected CHO cells replated with mock-transfected 7WD10 cells serves as the co-incubation condition, while mock-transfected CHO cells replated with ERdj3-transfected 7WD10 cells comprise the co-expression condition. Media inputs (1:150) are shown as a control.


Representative immunoblot of M2 anti - FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co - overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q . Media contained vehicle or trimethoprim ( TMP , 10 M , 16 h ; activates ATF6 ) , as indicated . Media inputs ( 1 : 400 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP - dependent activation of ATF6 . Bar graph depicting quantification of the change in the amount of ERdj3 co - immunoprecipitating with TTRA25T following activation of ATF6 , as in Figure 6A . The ERdj3 / TTR ratio is normalized to the ratio in the absence of ATF6 activation . * indicates a p - value < 0 . 05 ; n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co-overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q. Media contained vehicle or trimethoprim (TMP, 10 M, 16 h; activates ATF6), as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP-dependent activation of ATF6. Bar graph depicting quantification of the change in the amount of ERdj3 co-immunoprecipitating with TTRA25T following activation of ATF6, as in Figure 6A. The ERdj3/TTR ratio is normalized to the ratio in the absence of ATF6 activation. *indicates a p-value < 0.05; n = 3.


Representative immunoblot of M2 anti - FLAG immunopurification of media conditioned for 24 h on HEK293T cells co - overexpressing FTTTRA25T , ERdj3WT , ERdj3H53Q , and / or BiP , as indicated . Media inputs ( 1 : 400 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression .

Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293T cells co-overexpressing FTTTRA25T, ERdj3WT, ERdj3H53Q, and/or BiP, as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.


Quantification of immunoblots of M2 anti - FLAG immunopurification of media collected from cells co - overexpressing either ERdj3WT or ERdj3H53Q , FTTTRA25T , and BiP , as in Figure 6C . The ERdj3 / TTR ratio is normalized to the ratio in the absence of BiP overexpression . * * indicates a p - value < 0 . 01 ; n = 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblots of M2 anti-FLAG immunopurification of media collected from cells co-overexpressing either ERdj3WT or ERdj3H53Q, FTTTRA25T, and BiP, as in Figure 6C. The ERdj3/TTR ratio is normalized to the ratio in the absence of BiP overexpression. **indicates a p-value < 0.01; n = 5.


Immunoblot of 6E10 anti - APP immunopurifications from the conditioned media of CHO cells or CHO - derived APP751 - expressing ( 7WD10 ) cells overexpressing ERdj3WT , ERdj3H53Q , and BiP as indicated . Media inputs ( 1 : 100 ) are shown as a control . Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression .

Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT, ERdj3H53Q, and BiP as indicated. Media inputs (1:100) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.


( H ) Immunoblot representing levels mTOR - dependent targets with defined 5 ' TOP motifs 3 hours and 2 days after TAC . n = 3 biological replicates per condition .

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

(H) Immunoblot representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition.


( J ) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1 . * p ≤ 0 . 05 vs sham surgery , # p ≤ 0 . 05 vs TAC 2d surgery . t - test , n = 5 biological replicates per group . Data information : Error bars indicate means ±

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

(J) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM


( K ) RT - qPCR for indicated mRNAs 2 days after TAC for indicated groups , n = 5 biological replicates per group . Data information : Error bars indicate means ±

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

(K) RT-qPCR for indicated mRNAs 2 days after TAC for indicated groups, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM


( C ) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h , and 24h of Torin 1 treatment . mTORC1 induction was monitored by downstream targets , p4EBP1 and pS6K proteins . rpS5 - example of mTOR - dependent mRNA used as a positive control . Csq - calsequestrin used as a houskeeping protein .

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

(C) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h, and 24h of Torin 1 treatment. mTORC1 induction was monitored by downstream targets, p4EBP1 and pS6K proteins. rpS5 - example of mTOR-dependent mRNA used as a positive control. Csq - calsequestrin used as a houskeeping protein.


( F , G ) Ribo - seq and RNA - seq expression data of Cand2 in sham and TAC operated animals ( CPM - count per million ) . One - way ANOVA , * * - P ≤ 0 . 01 , n = 2 - 4 biological replicates per group . Data information : Error bars indicate means ±

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

(F, G) Ribo-seq and RNA-seq expression data of Cand2 in sham and TAC operated animals (CPM - count per million). One-way ANOVA, ** - P ≤ 0.01, n=2-4 biological replicates per group. Data information: Error bars indicate means ± SEM


( J ) Schematic representation of reporter consisting of human Cand2 5 ' UTR and downstream Renilla luciferase coding sequence . A potential TOP - like motif is highlighted in a grey box . Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites ( Release 10 . 1 ) . The effect of Cand2 5 ' UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1 . eEF2 - positive control of reporter with defined 5 ' TOP motif and non - TOP 5 ' UTR of β - actin used as a negative control . Total protein content in lysates used for luciferase assay . t - test , n = 4 - 5 biological replicates per group , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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

(J) Schematic representation of reporter consisting of human Cand2 5'UTR and downstream Renilla luciferase coding sequence. A potential TOP-like motif is highlighted in a grey box. Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites (Release 10.1). The effect of Cand2 5'UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1. eEF2 - positive control of reporter with defined 5'TOP motif and non-TOP 5'UTR of β-actin used as a negative control. Total protein content in lysates used for luciferase assay. t-test, n=4-5 biological replicates per group, ** - P ≤ 0.01, *** - P ≤ 0.001.


( A ) Representative immunoblot of Cand2 protein levels in mouse organs .

(A) Representative immunoblot of Cand2 protein levels in mouse organs.


( C ) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice . CAND2 ( stained in red ) , ACTIN ( stained in green ) and nuclei ( stained with DAPI , blue ) in . Scale bar

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

(C) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with DAPI, blue) in. Scale bar 20μm


( F ) Immunoblot of subcellular fractionation of left ventricle ( LV ) . Gapdh and Histone 3D ( H3D ) used as cytoplasmic and nuclear markers , respectively . WCL - whole cell lysate , cyt - cytoplasmic fraction , nuc - nuclear fraction .

O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of subcellular fractionation of left ventricle (LV). Gapdh and Histone 3D (H3D) used as cytoplasmic and nuclear markers, respectively. WCL - whole cell lysate, cyt - cytoplasmic fraction, nuc - nuclear fraction.


( E ) NRCMs size measurement after Cand2 OE and 24h PE stimulation . Analyzed by One - way ANOVA . n > 150 cells from n = 3 independent experiments . Violin plot : the middle horizontal lane - median , upper lane - 3rd quartile , lower lane - 1st quartile . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(E) NRCMs size measurement after Cand2 OE and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( F ) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT - qPCR . Analysed by One - way ANOVA . n = 5 - 9 biological replicates per conditions . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(F) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT-qPCR. Analysed by One-way ANOVA. n=5-9 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( J ) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT - qPCR . Analyzed by One - way ANOVA . n = 6 - 13 mice per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 . 0001

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

(J) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT-qPCR. Analyzed by One-way ANOVA. n=6-13 mice per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( B ) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6 . t - test , n = 4 - 5 biological replicates per group . Data information : Error bars indicate means ± SEM . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 .

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(B) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6. t-test, n=4-5 biological replicates per group. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.


( F ) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery . Lamin B - nuclear marker , Gapdh - cytoplasmic marker . Analyzed by One - way ANOVA , n = 3 biological replicates . Data information : Error bars indicate means ± SEM . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 .

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(F) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery. Lamin B - nuclear marker, Gapdh - cytoplasmic marker. Analyzed by One-way ANOVA, n=3 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.


( B ) mRNA levels of MEF2 - dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups . PE treatment for 24h ( 50uM ) . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(B) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups. PE treatment for 24h (50uM). Analyzed by One-way ANOVA, n=6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( E ) Grk5 protein and mRNA levels in NRCMs as well as MEF2 - luciferase activity after 24h of Torin 1 treatment . Analyzed by t - test , n = 4 - 6 biological replicates per conditions . Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein ( S235 / 236rpS6 ) . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(E) Grk5 protein and mRNA levels in NRCMs as well as MEF2-luciferase activity after 24h of Torin 1 treatment. Analyzed by t-test, n=4-6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6). Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( I ) Grk5 , Nppa , and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD . Analyzed by t - test , n = 3 biological replicates per conditions . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(I) Grk5, Nppa, and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD. Analyzed by t-test, n=3 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( B ) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2 . NRCMs were treated with MLN4924 ( 100 nM ) to block Cul1 neddylation . Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h . t - test , n = 4 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(B) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( D ) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition ( MLN ) analysed by immunoblot and RT - qPCR . Analyzed by One - way ANOVA , n = 4 - 5 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(D) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by immunoblot and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( H ) mRNA levels of MEF2 - dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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(H) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA, n= 6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( A ) Quantification of indicated mRNA levels 2 days after TAC measured by RT - qPCR . Analyzed by One - way ANOVA . n = 5 - 7 biological replicates per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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(A) Quantification of indicated mRNA levels 2 days after TAC measured by RT-qPCR. Analyzed by One-way ANOVA. n=5-7 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


( C ) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT - qPCR . Analyzed by One - way ANOVA . n = 3 - 5 biological replicates per group . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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(C) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT-qPCR. Analyzed by One-way ANOVA. n=3-5 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


( D ) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice . Analyzed by t - test . n = 5 - 8 mice per group . Cul1 neddylation was inhibited in C2C12 by MLN4924 ( - / + MLN ) for neddylated Cul1 band detection . Data information : Error bars indicate means ± SEM ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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(D) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice. Analyzed by t-test. n=5-8 mice per group. Cul1 neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


( B ) Box plots show the MIG - 6 ( ERRFI1 ) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas , analyzed using Illumina HumanWG - 6 _ v3 Arrays . The gene expression levels are determined using the R2 Genomics Analysis and Visualization Platform ( http : / / r2 . amc . nl ) . In the box plot , error bars are the 95 % confidence interval , the bottom and top of the box are the 25th and 75th percentiles , the line inside the box is the 50th percentile ( median ) , and any outliers are shown as open circles . * * p < 0 . 01 , by Student ' s t - test .

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(B) Box plots show the MIG-6 (ERRFI1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.


( C ) Immunoblotting analysis for MIG - 6 protein expression in various human breast cancer cell lines .

(C) Immunoblotting analysis for MIG-6 protein expression in various human breast cancer cell lines.


( D ) Representative images of histological analyses of MIG - 6 in TNBC patients in low - grade ( stage I ) and high - grade ( stage III ) tumors . Scale bar , 200 µm .

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(D) Representative images of histological analyses of MIG-6 in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors. Scale bar, 200 µm.


( E ) Quantification analysis of MIG - 6 expression in 85 cases of resected TNBCs . In the box plot , error bars are the 95 % confidence interval , the bottom and top of the box are the 25th and 75th percentiles , the line inside the box is the 50th percentile ( median ) . The p - value 0 . 011 is used for the comparison of all three groups , determined by the Kruskal - Wallis H test .

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(E) Quantification analysis of MIG-6 expression in 85 cases of resected TNBCs. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median). The p-value 0.011 is used for the comparison of all three groups, determined by the Kruskal-Wallis H test.


( F ) Kaplan - Meier plot analysis of the disease - specific survival of 85 TNBC breast cancer patients with low or high expression of MIG - 6 .

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(F) Kaplan-Meier plot analysis of the disease-specific survival of 85 TNBC breast cancer patients with low or high expression of MIG-6.


( G ) Kaplan - Meier plot analysis of the metastasis - specific survival of 85 TNBC patients with low or high expression of MIG - 6 . MIG - 6 protein expression in the 85 TNBC specimens was determined by H - score and widely distributed . MIG - 6 expression greater than or equal to the median is classified as " high , " while expression less than the median is classified as " low . "

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(G) Kaplan-Meier plot analysis of the metastasis-specific survival of 85 TNBC patients with low or high expression of MIG-6. MIG-6 protein expression in the 85 TNBC specimens was determined by H-score and widely distributed. MIG-6 expression greater than or equal to the median is classified as "high," while expression less than the median is classified as "low."


( A ) Cell proliferation and immunoblotting assays in BT549 cells with Luciferase or MIG - 6 knockdown .

(A) Cell proliferation and immunoblotting assays in BT549 cells with Luciferase or MIG-6 knockdown.


( B ) Cell proliferation and immunoblotting assays in MDA - MB - 231 cells with Luciferase or MIG - 6 knockdown .

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(B) Cell proliferation and immunoblotting assays in MDA-MB-231 cells with Luciferase or MIG-6 knockdown.


( C ) Representative images and quantification results for a colony formation assay in BT549 cells with GFP or MIG - 6 knockdown .

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(C) Representative images and quantification results for a colony formation assay in BT549 cells with GFP or MIG-6 knockdown.


( D Immunoblotting analysis for EGFR signaling in BT549 cells with GFP or MIG - 6 knockdown .

(D Immunoblotting analysis for EGFR signaling in BT549 cells with GFP or MIG-6 knockdown.


E ) Immunoblotting analysis for EGFR signaling in MDA - MB - 231 cells with GFP or MIG - 6 knockdown .

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E) Immunoblotting analysis for EGFR signaling in MDA-MB-231 cells with GFP or MIG-6 knockdown.


( F ) Cell growth assay in BT549 cells with GFP or MIG - 6 knockdown in the absence and presence of gefitinib for 72 hours .

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(F) Cell growth assay in BT549 cells with GFP or MIG-6 knockdown in the absence and presence of gefitinib for 72 hours.


( A ) Immunoblotting analysis for MIG - 6 expression in BT549 cells under normoxia and hypoxia .

(A) Immunoblotting analysis for MIG-6 expression in BT549 cells under normoxia and hypoxia.


( B ) Extracellular acidification rate ( ECAR ) in BT549 cells with GFP or MIG - 6 knockdown . The ECAR flux profile of GFP and MIG - 6 knockdown BT549 cells upon stimulation of glucose , oligomycin , and 2 - DG is shown on the left . Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG - 6 knockdown BT549 cells are indicated by orange and green boxes , graphed on the right . Results are presented as mean ± SEM ( n = 15 ; biological replicates ) .

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(B) Extracellular acidification rate (ECAR) in BT549 cells with GFP or MIG-6 knockdown. The ECAR flux profile of GFP and MIG-6 knockdown BT549 cells upon stimulation of glucose, oligomycin, and 2-DG is shown on the left. Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG-6 knockdown BT549 cells are indicated by orange and green boxes, graphed on the right. Results are presented as mean ± SEM (n=15; biological replicates).


( C ) A heatmap diagram shows the expression profile of genes involved in glycolysis , the TCA cycle , the pentose phosphate pathway , pyruvate oxidation , gluconeogenesis , and glycogen metabolism obtained from two sets of GFP and MIG - 6 knockdown BT549 cells .

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(C) A heatmap diagram shows the expression profile of genes involved in glycolysis, the TCA cycle, the pentose phosphate pathway, pyruvate oxidation, gluconeogenesis, and glycogen metabolism obtained from two sets of GFP and MIG-6 knockdown BT549 cells.


Histograms show the percentage of gene dysregulation in glycolysis , TCA cycle , upon MIG - 6 knockdown in BT549 cells . For each gene , the change was calculated based on the equation : ( shGFP - shMIG6 ) / shMIG - 6 . Results are presented as mean ± SEM ( n = 3 ; biological replicates ) .

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Histograms show the percentage of gene dysregulation in glycolysis, TCA cycle, upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).


F ) Histograms show the percentage of pyruvate oxidation pathways upon MIG - 6 knockdown in BT549 cells . For each gene , the change was calculated based on the equation : ( shGFP - shMIG6 ) / shMIG - 6 . Results are presented as mean ± SEM ( n = 3 ; biological replicates ) .

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F) Histograms show the percentage of pyruvate oxidation pathways upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).


( H ) Lactate production assay in BT549 cells with GFP or MIG - 6 knockdown ( n = 3 ) . The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer . The rate of lactate production was determined ( lactate production rate = lactate concentration / cells / time ) and normalized to the rate detected in the control group . The quantified results are presented as mean ± SEM ( n = 3 ; biological replicates ) .

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(H) Lactate production assay in BT549 cells with GFP or MIG-6 knockdown (n=3). The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer. The rate of lactate production was determined (lactate production rate = lactate concentration/cells/time) and normalized to the rate detected in the control group. The quantified results are presented as mean ± SEM (n=3; biological replicates).


( A ) Box plots show the GLUT1 ( SLC2A1 ) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas , analyzed using Illumina HumanWG - 6 _ v3 Arrays . The gene expression levels are determined using the R2 platform . In the box plot , error bars are the 95 % confidence interval , the bottom and top of the box are the 25th and 75th percentiles , the line inside the box is the 50th percentile ( median ) , and any outliers are shown as open circles . * * p < 0 . 01 , by Student ' s t - test . .

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(A) Box plots show the GLUT1 (SLC2A1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 platform. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test..


( B ) Real - Time PCR analysis for GLUT1 and MIG - 6 mRNA expression in GFP and MIG - 6 knockdown BT549 cells ( n = 3 ) .

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(B) Real-Time PCR analysis for GLUT1 and MIG-6 mRNA expression in GFP and MIG-6 knockdown BT549 cells (n=3).


( C ) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG - 6 knockdown .

(C) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG-6 knockdown.


( D ) Confocal image analysis results for membrane - bound GLUT1 in GFP and MIG - 6 knockdown BT549 cells are shown in the top panel . The arrow indicates membrane - localized GLUT1 . Scale bar , 25 μm . Quantitative analysis results of the percentage of GFP and MIG - 6 knockdown BT549 cells expressing membrane - localized GLUT1 are shown in the bottom panel ( n = 3 ) .

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(D) Confocal image analysis results for membrane-bound GLUT1 in GFP and MIG-6 knockdown BT549 cells are shown in the top panel. The arrow indicates membrane-localized GLUT1. Scale bar, 25 μm. Quantitative analysis results of the percentage of GFP and MIG-6 knockdown BT549 cells expressing membrane-localized GLUT1 are shown in the bottom panel (n=3).


( E ) Representative flow cytometry images ( upper ) and quantitative results ( lower ) of measuring glucose uptake in GFP and MIG - 6 knockdown BT549 cells ( n = 3 ) . Cells were grown in the presence of the fluorescent analog 2 - NBDG for various time periods , and glucose uptake was quantified using flow cytometric analysis . The red box indicates the gating for 2 - NBDG + cells and the number indicates the percentage of 2 - NBDG + cells .

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(E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells.


( F ) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG - 6 knockdown BT549 cells with or without MG132 treatment .

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(F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment.


( B ) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG - 6 knockdown . ( C ) Immunoblotting analysis for HIF1α protein expression in MDA - MB - 231 cells with GFP or MIG - 6 knockdown .

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(B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown. (C) Immunoblotting analysis for HIF1α protein expression in MDA-MB-231 cells with GFP or MIG-6 knockdown.


( B ) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG - 6 knockdown .

(B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown.


( D ) Real - Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG - 6 knockdown . The quantified results are presented as mean ± SD ( n = 4 ) . * * * p < 0 . 001 , by Student ' s t - test .

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(D) Real-Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG-6 knockdown. The quantified results are presented as mean ± SD (n=4). ***p < 0.001, by Student's t-test.


( E ) Immunoblotting analysis for HIF1α protein expression in GFP and MIG - 6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132 .

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(E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132.


( F ) GFP and MIG - 6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132 , subjected to immunoprecipitation ( IP ) with Flag - tag or HIF1α antibody , followed by immunoblotting analysis . MG132 was used to rescue HIF1α protein degradation mediated by MIG - 6 knockdown , leading to similar HIF1α protein levels in control and MIG - 6 knockdown BT549 cells , which were used as the input to examine the role of MIG - 6 in the interaction between HIF1α and HAUSP .

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(F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP.


( G ) Luciferase and MIG - 6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment . Afterward , cells were harvested for IP with HA antibody , followed by immunoblotting analysis to determine the level of K48 - linked ubiquitination of HIF1α . MG132 was used to preserve the degradative K48 - linked ubiquitination signals .

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

(G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals.


( H ) Immunoblotting analysis for MIG - 6 and HIF1α expression in Luciferase and MIG - 6 knockdown 293 without MG132 treatment .

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

(H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment.


( I ) Immunoblotting analysis for GLUT1 protein expression upon MIG - 6 overexpression in GFP and HIF1α knockdown BT549 cells .

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

(I) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HIF1α knockdown BT549 cells.


( J ) Immunoblotting analysis for GLUT1 protein expression upon MIG - 6 overexpression in GFP and HAUSP knockdown BT549 cells .

(J) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HAUSP knockdown BT549 cells.


( K ) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG - 6 knockdown BT549 cells .

(K) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG-6 knockdown BT549 cells.


( A and B ) Lactate production assay in BT549 ( A ) and MDA - MB - 231 ( B ) cells with Luciferase or GLUT1 knockdown .

O O O O O 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 and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown.


( C ) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown .

(C) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown.


( D ) Cell proliferation assay in MDA - MB - 231 cells with GFP or GLUT1 knockdown .

(D) Cell proliferation assay in MDA-MB-231 cells with GFP or GLUT1 knockdown.


( E and F ) Immunoblotting analysis for GLUT1 protein expression in control and MIG - 6 knockdown BT549 ( E ) and MDA - MB - 231 ( F ) cells with or without GLUT1 overexpression .

O O O O O O O O O O O O O O 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 and F) Immunoblotting analysis for GLUT1 protein expression in control and MIG-6 knockdown BT549 (E) and MDA-MB-231 (F) cells with or without GLUT1 overexpression.


( G Lactate production assay in control and MIG - 6 knockdown BT549 cells with or without GLUT1 overexpression .

(G Lactate production assay in control and MIG-6 knockdown BT549 cells with or without GLUT1 overexpression.


H ) Lactate production assay in control and MIG - 6 knockdown MDA - MB - 231 cells with or without GLUT1 overexpression .

H) Lactate production assay in control and MIG-6 knockdown MDA-MB-231 cells with or without GLUT1 overexpression.


( I and J ) Cell proliferation assay in BT549 ( I ) and MDA - MB - 231 ( J ) cells with control knockdown , MIG - 6 knockdown , or MIG - 6 knockdown plus GLUT1 overexpression .

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

(I and J) Cell proliferation assay in BT549 (I) and MDA-MB-231 (J) cells with control knockdown, MIG-6 knockdown, or MIG-6 knockdown plus GLUT1 overexpression.


( A and B ) In vivo primary tumor growth derived from BT549 cells with Luciferase or MIG - 6 knockdown ( six mice per group ) . Cells were injected into the mammary fat pads of nude mice , and tumor sizes were measured weekly by caliper . Kaplan - Meier plot analysis is used to determine the incidence of Luciferase or MIG - 6 knockdown BT549 - xenograft tumors ( A ) . Volumes of Luciferase or MIG - 6 knockdown BT549 tumors at week 10 are presented as mean ± SEM ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) In vivo primary tumor growth derived from BT549 cells with Luciferase or MIG-6 knockdown (six mice per group). Cells were injected into the mammary fat pads of nude mice, and tumor sizes were measured weekly by caliper. Kaplan-Meier plot analysis is used to determine the incidence of Luciferase or MIG-6 knockdown BT549-xenograft tumors (A). Volumes of Luciferase or MIG-6 knockdown BT549 tumors at week 10 are presented as mean ± SEM (B).


( C In vivo primary tumor growth derived from MDA - MB - 231 cells with Luciferase or MIG - 6 knockdown ( nine mice per group ) . Volumes of Luciferase or MIG - 6 knockdown MDA - MB - 231 tumors were measured weekly by caliper are presented as mean ± SEM

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vivo primary tumor growth derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Volumes of Luciferase or MIG-6 knockdown MDA-MB-231 tumors were measured weekly by caliper are presented as mean ± SEM


( C In vivo primary tumor growth derived from MDA - MB - 231 cells with Luciferase or MIG - 6 knockdown ( nine mice per group ) . Volumes of Luciferase or MIG - 6 knockdown MDA - MB - 231 tumors were measured weekly by caliper are presented as mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) In vivo primary tumor derived from MDA - MB - 231 cells with Luciferase or MIG - 6 knockdown ( nine mice per group ) . Tumor weights of MDA - MB - 231 - derived xenografts were measured at the endpoint ( day 33 ) and are presented as mean ± SEM ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo primary tumor derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Tumor weights of MDA-MB-231-derived xenografts were measured at the endpoint (day 33) and are presented as mean ± SEM (D).


( E ) Immunoblotting analysis for MIG - 6 expression in BT549 cells with MIG - 6 inducible knockdown ( iMIG - 6 - shRNA ) and the non - targeting shRNA control ( iNT - shRNA ) upon doxycycline treatment .

O O O O O O O O O O O O O O O O O O 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) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment.


( F ) In vivo primary tumor growth derived from BT549 cells containing iNT - shRNA and iMIG - 6 - shRNA ( n = 7 per group ) . The arrow indicates the time point when the diet was given to induce MIG - 6 knockdown in vivo . Volumes of iNT - shRNA and iMIG - 6 - shRNA BT549 tumors are presented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo primary tumor growth derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA (n=7 per group). The arrow indicates the time point when the diet was given to induce MIG-6 knockdown in vivo. Volumes of iNT-shRNA and iMIG-6-shRNA BT549 tumors are presented as mean ± SEM.


( G ) Representative images for tumor growth and quantitative results for tumor weight at the experimental endpoint ( seven mice per group ) . Results are presented as mean ± SEM .

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

(G) Representative images for tumor growth and quantitative results for tumor weight at the experimental endpoint (seven mice per group). Results are presented as mean ± SEM.


( A ) Representative images of histological analysis for GLUT1 protein expression in BT549 - derived xenograft tumors . Scale bar , 100 μm . ( B ) Representative images of histological analysis for GLUT1 protein expression in xenograft tumors derived from BT549 cells containing iNT - shRNA and iMIG - 6 - shRNA . 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 O O O O O O O O O O O O O O O O O O O

(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm. (B) Representative images of histological analysis for GLUT1 protein expression in xenograft tumors derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA. Scale bar, 100 μm.


( A ) Representative images of histological analysis for GLUT1 protein expression in BT549 - derived xenograft tumors . 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

(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm.


( C ) Representative images of histological analyses for GLUT1 and MIG - 6 expressions in TNBC patients in low - grade ( stage I ) and high - grade ( stage III ) tumors Scale bar , 200 µm .

(C) Representative images of histological analyses for GLUT1 and MIG-6 expressions in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors Scale bar, 200 µm.


( D ) Scatterplot analysis for the correlation between GLUT1 and MIG - 6 expression levels ( n = 85 ) . The r value was calculated by Spearman ' s rank correlation coefficient analysis .

O O O O O O O O O O O O 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) Scatterplot analysis for the correlation between GLUT1 and MIG-6 expression levels (n=85). The r value was calculated by Spearman's rank correlation coefficient analysis.


( E ) Representative images of the immunofluorescence multiplex assay for colocalization of MIG - 6 and HIF1α protein expression in TNBC tumor specimens . The triangle indicates nuclear colocalization , and the star indicates cytoplasmic colocalization .

(E) Representative images of the immunofluorescence multiplex assay for colocalization of MIG-6 and HIF1α protein expression in TNBC tumor specimens. The triangle indicates nuclear colocalization, and the star indicates cytoplasmic colocalization.


Lymphocyte subsets from 12 month old Ptpn2fl / fl ; p53 + / - and Lck - Cre ; Ptpn2fl / f ; p53 + / - mice were analyzed by flow cytometry . Significance in ( a ) was determined using two - sided Fisher ' s exact test .

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

Lymphocyte subsets from 12 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were analyzed by flow cytometry. Significance in (a) was determined using two-sided Fisher's exact test.


AT - 3 - OVA breast cancer cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl / fl and Lck - Cre ; Ptpn2fl / fl mice and ( c ) tumour growth monitored over 26 days .

AT-3-OVA breast cancer cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice and (c) tumour growth monitored over 26 days.


At day 26 ( d26 ) the numbers of activated tumour infiltrating lymphocytes ( TILs ) were determined .

At day 26 (d26) the numbers of activated tumour infiltrating lymphocytes (TILs) were determined.


The proportion of IFNγ + versus IFNγ + TNF + d26 TILs was determined by flow cytometry .

The proportion of IFNγ+ versus IFNγ+TNF+ d26 TILs was determined by flow cytometry.


d26 TILS were incubated with AT - 3 - OVA tumour cells isolated from tumour bearing C57BL / 6 mice and the proportion of IFNγ + T cells was determined .

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

d26 TILS were incubated with AT-3-OVA tumour cells isolated from tumour bearing C57BL/6 mice and the proportion of IFNγ+ T cells was determined.


AT - 3 - OVA mammary tumour cells ( 1x106 ) were injected into the fourth inguinal mammary fat pads of female Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T cells from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . Tumour - bearing Ly5 . 1 + mice were monitored for a ) tumour growth over 21

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. Tumour-bearing Ly5.1+ mice were monitored for a) tumour growth over 21 days


AT - 3 - OVA mammary tumour cells ( 1x106 ) were injected into the fourth inguinal mammary fat pads of female Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T cells from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . After 21 days TILs were processed for flow cytometry and donor T cell numbers ( Ly5 . 1 - Ly5 . 2 + ) determined .

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 21 days TILs were processed for flow cytometry and donor T cell numbers (Ly5.1-Ly5.2+) determined.


AT - 3 - OVA mammary tumour cells ( 1x106 ) were injected into the fourth inguinal mammary fat pads of female Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T cells from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . After 9 days the proportion of Ly5 . 2 + IFNγ + TNF + versus Ly5 . 2 + GrzB + TILs was determined .

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 9 days the proportion of Ly5.2+IFNγ+TNF+ versus Ly5.2+GrzB+ TILs was determined.


AT - 3 - OVA mammary tumour cells ( 1x106 ) were injected into the fourth inguinal mammary fat pads of female Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T cells from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . d ) for survival over 86 days .

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. d) for survival over 86 days.


Gene expression in tumours from mice treated with Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl T cells 21 days post - adoptive transfer versus those re - emerging in mice treated with Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl T cells .

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

Gene expression in tumours from mice treated with Ly5.2+;OT-1;Ptpn2fl/fl T cells 21 days post-adoptive transfer versus those re-emerging in mice treated with Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl T cells.


B16 . F10 - OVA melanoma cells ( 1x105 ) were engrafted onto the abraded skin in the flanks of Ly5 . 1 + mice . 24 h after tumour cell engraftment naïve CD8 + CD62LhiCD44lo lymph node T cells from Ly5 . 2 + OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred and tumour incidence monitored .

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

B16.F10-OVA melanoma cells (1x105) were engrafted onto the abraded skin in the flanks of Ly5.1+ mice. 24 h after tumour cell engraftment naïve CD8+CD62LhiCD44lo lymph node T cells from Ly5.2+ OT-1;Ptpn2fl/fl versus Ly5.2+ OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred and tumour incidence monitored.


Epidermal lymphocytes from tumour - free mice were stained for CD69hiCD103hi and donor - derived ( Ly5 . 2 + vα2 + ) tissue resident memory T cells ( TRMs ) determined by flow cytometry .

Epidermal lymphocytes from tumour-free mice were stained for CD69hiCD103hi and donor-derived (Ly5.2+vα2+) tissue resident memory T cells (TRMs) determined by flow cytometry.


Tumour sizes in B16 . F10 - OVA melanoma bearing mice were determined between day 21 and 23 .

Tumour sizes in B16.F10-OVA melanoma bearing mice were determined between day 21 and 23.


TILs were assessed for Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl and Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl donor T cell numbers by flow cytometry .

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

TILs were assessed for Ly5.2+;OT-1;Ptpn2fl/fl and Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl donor T cell numbers by flow cytometry.


CD8 + HER - 2 CAR - T cells generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes were stained for intracellular p ( Y418 ) - SFK and p ( Y418 ) - SFK MFIs were determined by flow cytometry .

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

CD8+ HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes were stained for intracellular p(Y418)-SFK and p(Y418)-SFK MFIs were determined by flow cytometry.


HER - 2 - specific Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - CAR - T cells were incubated with HER - 2 expressing 24JK sarcoma cells ( 24JK - HER - 2 ) or HER - 2 negative 24JK sarcoma cells and CD44 , CD25 , PD - 1 and LAG - 3 MFIs on CD8 + CAR - T cells were determined by flow cytometry .

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

HER-2-specific Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with HER-2 expressing 24JK sarcoma cells (24JK-HER-2) or HER-2 negative 24JK sarcoma cells and CD44, CD25, PD-1 and LAG-3 MFIs on CD8+ CAR-T cells were determined by flow cytometry.


Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl , or Lck - Cre ; Ptpn2fl / fl ; Lck + / - HER - 2 CAR - T cells were incubated with 24JK - HER - 2 or 24JK sarcoma cells and the proportion of CD8 + IFNγ + versus CD8 + IFNγ + TNF + CAR - T cells determined by flow cytometry .

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

Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and the proportion of CD8+IFNγ+ versus CD8+IFNγ+TNF+ CAR-T cells determined by flow cytometry.


Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl or Lck - Cre ; Ptpn2fl / fl ; Lck + / - HER - 2 CAR - T cells were incubated with 5 μM CTV - labelled ( CTVbright ) 24JK - HER - 2 and 0 . 5 μM CTV - labelled ( CTVdim ) 24JK sarcoma cells . Antigen - specific target cell lysis was monitored for the depletion of CTVbright 24JK - HER - 2 cells by flow cytometry .

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

Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 5 μM CTV-labelled (CTVbright) 24JK-HER-2 and 0.5 μM CTV-labelled (CTVdim) 24JK sarcoma cells. Antigen-specific target cell lysis was monitored for the depletion of CTVbright 24JK-HER-2 cells by flow cytometry.


HER - 2 - specific Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl and Lck - Cre ; Ptpn2fl / fl ; Lck + / - CAR - T cells were incubated with plate - bound α - CD3 and CD25 MFIs on CD8 + CD44hiCD62Llo versus CD8 + CD44hiCD62Lhi CAR - T cells determined by flow cytometry .

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

HER-2-specific Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with plate-bound α-CD3 and CD25 MFIs on CD8+CD44hiCD62Llo versus CD8+CD44hiCD62Lhi CAR-T cells determined by flow cytometry.


HER - 2 - E0771 mammary tumour cells ( 2x105 ) were injected into the fourth inguinal mammary fat pads of female HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total body irradiation ( 4 Gy ) followed by the adoptive transfer of 6x106 FACS - purified CD8 + CD44hiCD62Lhi central memory HER - 2 CAR - T cells generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl or Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T cell transfer and monitored for tumour growth .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl or Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.


TILs isolated from HER - 2 - E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for c ) CD11b + F4 / 80hiLy6ChiLy6Glo myeloid derived suppressor cells ( MDSC ) by flow cytometry .

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

TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for c) CD11b+ F4/80hiLy6ChiLy6Glo myeloid derived suppressor cells (MDSC) by flow cytometry.