Figure 4Immunoblotting of ATF4 in melanoma cell lines ( A ) treated with the LDHA inhibitor LDHAi for 96 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Immunoblotting of ATF4 in melanoma cell lines ( B ) transfected with non - targeting ( NT ) or LDHA - specific siRNAs for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector ( EV ) or indicated sh - LDHAs . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . D Immunoblotting of ATF4 , c - MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2 , 24 , or 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock - or si - ATF4 - treated for 24 hr and then incubated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . F qRT - PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si - ATF4 for 24 hr and then incubated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular essential amino acid levels in MeWo cells treated and share Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001H Proliferation of melanoma cell lines mock - or si - ATF4 - transfected and incubated with LDHAi as indicated for up to 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , and two - way Anova was used for the proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001qRT - PCR analysis of asparagine synthetase ( ASNS ) and glutamine - pyruvate transaminase 2 ( GPT2 ) mRNA levels in the indicated cell lines after 24 hr treatment with LDHAi . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular asparagine ( Asn ) and alanine ( Ala ) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular Asn and Ala levels in MeWo cells mock - transfected or transfected with si - ATF4 ( K ) for 24 hr and then treated with LDHAi for 48 hr . and shared Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular Asn and Ala levels in MeWo cells mock - transfected or si - SLC1A5 ( L ) for 24 hr and then treated with LDHAi for 48 hr . and shared Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001M ( left ) Immunoblotting of ASNS in MeWo cells mock - or si - ASNS - transfected for 24 hr and then incubated with LDHAi for 48 hr . ( right ) Proliferation of MeWo cells mock - or si - ASNS - transfected for 24 hr and then incubated with LDHAi for the indicated times . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , and two - way Anova was used for the proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001N Immunoblotting of GCN2 , ATF4 and SLC1A5 in MeWo cells mock - or si - GCN2 - treated for 24 hr and then incubated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . O 3H - Glutamine uptake in MeWo cells transfected with si - GCN2 and treated 48 hr later with LDHAi for 6 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001P Gln levels in the medium after treatment of MeWo cells with mock - or si - GCN2 for 24 hr and subsequent incubation with LDHA - i for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001Q GC - MS - based quantification of intracellular Gln and essential amino acid levels in MeWo cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunoblotting of ATF4 in melanoma cell lines (A) treated with the LDHA inhibitor LDHAi for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.Immunoblotting of ATF4 in melanoma cell lines (B) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector (EV) or indicated sh-LDHAs. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.D Immunoblotting of ATF4, c-MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2, 24, or 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock- or si-ATF4-treated for 24 hr and then incubated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.F qRT-PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si-ATF4 for 24 hr and then incubated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated and share Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001H Proliferation of melanoma cell lines mock- or si-ATF4-transfected and incubated with LDHAi as indicated for up to 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001qRT-PCR analysis of asparagine synthetase (ASNS) and glutamine-pyruvate transaminase 2 (GPT2) mRNA levels in the indicated cell lines after 24 hr treatment with LDHAi. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular asparagine (Asn) and alanine (Ala) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or transfected with si-ATF4 (K) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or si-SLC1A5 (L) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001M (left) Immunoblotting of ASNS in MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for 48 hr. (right) Proliferation of MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for the indicated times. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001N Immunoblotting of GCN2, ATF4 and SLC1A5 in MeWo cells mock- or si-GCN2-treated for 24 hr and then incubated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.O 3H-Glutamine uptake in MeWo cells transfected with si-GCN2 and treated 48 hr later with LDHAi for 6 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001P Gln levels in the medium after treatment of MeWo cells with mock- or si-GCN2 for 24 hr and subsequent incubation with LDHA-i for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001Q GC-MS-based quantification of intracellular Gln and essential amino acid levels in MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Figure 5A GC - MS - based quantification of intracellular Asp ( upper ) and Ser ( lower ) levels in melanoma cell lines treated with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001B ( upper ) GC - MS - based 13C6 - glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi . ( lower ) GC - MS - based 13C5 - glutamine fractional isotope labeling of Asp in melanoma cell lines treated for 72 hr with LDHAi . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM ( C ) and treated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular Ser and Asp levels in MeWo cells cultured in MEM supplemented with Ser ( 400 μM ; D ) and treated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - MS - based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001F GC - MS - based quantification of intracellular Asp levels in MeWo cells pre - treated with Asp ( 10 mM ) for 6 hr and then treated with LDHAi for 1 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001G Immunoblotting of ATF4 in MeWo cells pre - treated with Asp ( 10 mM ) for 6 hr and then treated with GSK - 2837808A ( 10 μM ) for 1 , 2 , or 24 hr . H Immunoblotting of ATF4 in MeWo cells pre - treated with Ser ( 400 μM ) , Asp ( 10 mM ) , or Ser ( 400 μM ) + Asp ( 10 mM ) for 6 hr and then treated for 1 hr with LDHAi ( 10 μM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A GC-MS-based quantification of intracellular Asp (upper) and Ser (lower) levels in melanoma cell lines treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001B (upper) GC-MS-based 13C6-glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi. (lower) GC-MS-based 13C5-glutamine fractional isotope labeling of Asp in melanoma cell lines treated for 72 hr with LDHAi. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM (C) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in MEM supplemented with Ser (400 μM; D) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001F GC-MS-based quantification of intracellular Asp levels in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001G Immunoblotting of ATF4 in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with GSK-2837808A (10 μM) for 1, 2, or 24 hr.H Immunoblotting of ATF4 in MeWo cells pre-treated with Ser (400 μM), Asp (10 mM), or Ser (400 μM) + Asp (10 mM) for 6 hr and then treated for 1 hr with LDHAi (10 μM).


Figure 6A Relative NAD + / NADH ratio in MeWo cells treated with LDHAi , nicotinamide mononucleotide ( NMN , 1 mM ) , or a combination of the two for 24 hr . Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001B GC - MS - based quantification of total intracellular levels of Ser and Asp and their 13C6 - glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide ( NMN , 1 mM ) for 6 hr and then treated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001D GC - MS - based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001E GC - MS - based 13C6 - Glc ( M + 2 ) and 13C5 - Gln ( M + 4 ) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo cells treated with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001F GC - MS - based 13C6 - Glc ( left ) and 13C5 - Gln ( right ) isotopologue levels of Asp in MeWo cells treated with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001G GC - MS - based quantification of intracellular Ala ( left ) and Asp ( right ) levels in si - GPT2 - treated MeWo cells incubated with LDHAi for 1 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups and unpaired t - test for the comparison of two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001H Immunoblotting of ATF4 and GPT2 in MeWo

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O

Figure 6A Relative NAD+/NADH ratio in MeWo cells treated with LDHAi, nicotinamide mononucleotide (NMN, 1 mM), or a combination of the two for 24 hr. Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001B GC-MS-based quantification of total intracellular levels of Ser and Asp and their 13C6-glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide (NMN, 1 mM) for 6 hr and then treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001D GC-MS-based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001E GC-MS-based 13C6-Glc (M+2) and 13C5-Gln (M+4) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001F GC-MS-based 13C6-Glc (left) and 13C5-Gln (right) isotopologue levels of Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001G GC-MS-based quantification of intracellular Ala (left) and Asp (right) levels in si-GPT2-treated MeWo cells incubated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001H Immunoblotting of ATF4 and GPT2 in MeWo cells


Figure 7A ( left ) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible ( Tet - On ) sh - LDHA expression vector treated with doxycycline ( Dox ) for 48 hr . ( right ) qRT - PCR ( n = 3 ) analysis of the ATF4 target genes in A375 cells treated with doxycycline ( 0 . 5 μg / ml ) for 24 hr . Statistical analysis was performed by unpaired t - test . Data are shown as the mean ± SEM , ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . B Proliferation of inducible ( Tet - On ) sh - LDHA expression vector harboring A375 cells ( n = 3 ) treated with Dox ( 0 . 5 μgC Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible ( Tet - On ) sh - LDHA expression vector . Starting day 5 , animals were treated on alternate days with 1 g / L doxycycline , 4 mg / kg rapamycin , or with a combination of the two ( n = 8 in each group ) . The tumor growth was determined by measuring the tumor volume . Statistical analysis was performed using Welch ' s t - test ( two - tailed ) . Data are shown as the mean ± SEM ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . D Immunoblotting of resected tumors ( 4 from each group ) for the indicated proteins . Representative images of H & E and cleaved caspase - 3 staining from each treatment group ( E ) . Quantification of cleaved caspase - 3 ( F ) ( n = 4 , each 4 fields ) . Scale bars , 50 μm . Statistical analysis was performed using two - way Anova . Data are shown as the mean ± SEM , ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . G Relative mTORC1 expression in pan - tumor samples segregated based on high ( LDHAhi ) ( n = 2917 ) and low LDHA ( LDHAlo ) ( n = 2914 ) expression . The p - value of paired Wilcoxon test following the adjustment multiple hypothesis correction ( FDR ) . Data information : In the boxplots , the top and bottom horizontal lines represent the 75th and the 25th percentile respectively , and the middle horizontal line represents the median . The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively . H Conditional essentiality of the indicated genes in LDHAlo and LDHAhi cell lines ( n = 17 , each group ) . The p - value of paired Wilcoxon test following the adjustment multiple hypothesis correction ( FDR ) . p ≤ 0 . 1 is significant . Data information : In the boxplots , the top and bottom horizontal lines represent the 75th and the 25th percentile respectively , and the middle horizontal line represents the median . The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A (left) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible (Tet-On) sh-LDHA expression vector treated with doxycycline (Dox) for 48 hr. (right) qRT-PCR (n=3) analysis of the ATF4 target genes in A375 cells treated with doxycycline (0.5 μg/ml) for 24 hr. Statistical analysis was performed by unpaired t-test. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.B Proliferation of inducible (Tet-On) sh-LDHA expression vector harboring A375 cells (n=3) treated with Dox (0.5 μgC Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible (Tet-On) sh-LDHA expression vector. Starting day 5, animals were treated on alternate days with 1 g/L doxycycline, 4 mg/kg rapamycin, or with a combination of the two (n=8 in each group). The tumor growth was determined by measuring the tumor volume. Statistical analysis was performed using Welch's t-test (two-tailed). Data are shown as the mean ± SEM ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.D Immunoblotting of resected tumors (4 from each group) for the indicated proteins.Representative images of H&E and cleaved caspase-3 staining from each treatment group (E).Quantification of cleaved caspase-3 (F) (n = 4, each 4 fields). Scale bars, 50 μm. Statistical analysis was performed using two-way Anova. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.G Relative mTORC1 expression in pan-tumor samples segregated based on high (LDHAhi) (n=2917) and low LDHA (LDHAlo) (n=2914) expression. The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively.H Conditional essentiality of the indicated genes in LDHAlo and LDHAhi cell lines (n=17, each group). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). p≤0.1 is significant. Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively.


Figure 8A qRT - PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor ( PLX - 4032 , 1 μM ) for 24 hr . Data information : Statistical analysis was performed by unpaired t - test for the comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . GC - MS - based quantification of ( B ) intracellular 3 - PG in A375 cells treated with PLX - 4032 ( 1 μM ) for 16 hr . Data information : Statistical analysis was performed by unpaired t - test for the comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . GC - MS - based quantification of ( C ) lactate levels in A375 cells treated with PLX - 4032 ( 1 μM ) for 16 hr . Data information : Statistical analysis was performed by unpaired t - test for the comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . D Immunoblotting of the indicated proteins in A375 cells treated with PLX - 4032 ( 1 μM ) for 2 , 6 , or 24 hr . E , F Heatmap for expression fold - change of ASNS , GPT2 , PSAT1 , and MITF between ( E ) post - treatment ( BRAF inhibitor , BRAF - i ; MEK inhibitor , MEK - i ) and pre - treatment , or ( F ) post - treatment ( BRAF - i ) and pre - treatment tumor in each of the patients ( y - axis ) . The p - value of paired Wilcoxon test following the adjustment multiple hypothesis correction ( FDR ) are also displayed ; post - treatment expression significantly greater than pre - treatment except in the case of MITF , where post - treatment expression is significantly less than pre - treatment .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A qRT-PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor (PLX-4032, 1 μM) for 24 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.GC-MS-based quantification of (B) intracellular 3-PG in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.GC-MS-based quantification of (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.E, F Heatmap for expression fold-change of ASNS, GPT2, PSAT1, and MITF between (E) post-treatment (BRAF inhibitor, BRAF-i; MEK inhibitor, MEK-i) and pre-treatment, or (F) post-treatment (BRAF-i) and pre-treatment tumor in each of the patients (y-axis). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR) are also displayed; post-treatment expression significantly greater than pre-treatment except in the case of MITF, where post-treatment expression is significantly less than pre-treatment.


figf1 ( a ) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6 . 0 array reveals a correlated pattern of variation in intensity across six copy - number probes spanning the region upstream of IRGM , suggesting the existence of a common copy - number polymorphism . ( Darker shades of red represent reduced hybridization intensity . ) Quantitative PCR indicated that the copy - number - variable region was an insertion / deletion ( Supplementary Fig . 1 ) . ( b ) Mapping of the deletion breakpoints by microarray and PCR . Red indicates evidence for deletion ; black indicates evidence to the contrary ; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele . ( d ) CD association of typed and imputed polymorphisms in the IRGM region in the NIDDK IBDGC genome scan . Blue trace indicates recombination map . The deletion polymorphism and rs13361189 are indicated by red arrows . rs13361189 ( which is in perfect linkage disequilibrium with the deletion , r2 = 1 . 0 ) was also the most strongly associated SNP in the combined WTCCC genome scan and replication study .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6.0 array reveals a correlated pattern of variation in intensity across six copy-number probes spanning the region upstream of IRGM, suggesting the existence of a common copy-number polymorphism. (Darker shades of red represent reduced hybridization intensity.) Quantitative PCR indicated that the copy-number-variable region was an insertion/deletion (Supplementary Fig. 1).(b) Mapping of the deletion breakpoints by microarray and PCR. Red indicates evidence for deletion; black indicates evidence to the contrary; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele.(d) CD association of typed and imputed polymorphisms in the IRGM region in the NIDDK IBDGC genome scan. Blue trace indicates recombination map. The deletion polymorphism and rs13361189 are indicated by red arrows. rs13361189 (which is in perfect linkage disequilibrium with the deletion, r2 = 1.0) was also the most strongly associated SNP in the combined WTCCC genome scan and replication study.


figf2 ( a ) The exonic SNP rs10065172 , which is in strong linkage disequilibrium with the 20 - kb insertion / deletion polymorphism upstream of IRGM , can be used to distinguish IRGM transcripts that arise from the deletion haplotype from IRGM transcripts that arise from the reference haplotype . This makes it possible to measure the relative expression of the two haplotypes in heterozygotes by measuring the relative abundance of the two rs10065172 alleles in cDNA . ( Yellow and green rectangles indicate transcribed sequence ; green rectangles indicate protein - coding sequence . ) ( b - g ) Differential expression of IRGM from the two structural haplotypes in heterozygotes . The relative abundance of the C and T alleles of rs10065172 in cDNA ( colored circles ) and genomic DNA ( black squares ) from human tissues and cell lines were measured . Gray squares indicate control measurements for genomic DNA from 48 HapMap YRI individuals , identifying the three reference genotype classes CC , CT and TT . Genomic DNA from all other samples was heterozygous for the C and T alleles ( black squares ) ; HeLa genomic DNA ( b , c ) showed a 2 : 1 allelic ratio ( CCT ) reflecting HeLa ' s triploid 5q karyotypic status . In e , cDNA and genomic DNA from lymphoblastoid cell lines from ten different heterozygous individuals were analyzed : the ten different colors represent the ten individuals ; technical replicates from the same cell line are connected by line segments . HeLa , SNU182 and the lymphoblastoid cell lines expressed the C allele of rs10065172 more strongly than the T allele ( b - e ) ; HCT116 and primary bronchial cells expressed the T allele much more strongly than the C allele ( f , g ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) The exonic SNP rs10065172, which is in strong linkage disequilibrium with the 20-kb insertion/deletion polymorphism upstream of IRGM, can be used to distinguish IRGM transcripts that arise from the deletion haplotype from IRGM transcripts that arise from the reference haplotype. This makes it possible to measure the relative expression of the two haplotypes in heterozygotes by measuring the relative abundance of the two rs10065172 alleles in cDNA. (Yellow and green rectangles indicate transcribed sequence; green rectangles indicate protein-coding sequence.) (b-g) Differential expression of IRGM from the two structural haplotypes in heterozygotes. The relative abundance of the C and T alleles of rs10065172 in cDNA (colored circles) and genomic DNA (black squares) from human tissues and cell lines were measured. Gray squares indicate control measurements for genomic DNA from 48 HapMap YRI individuals, identifying the three reference genotype classes CC, CT and TT. Genomic DNA from all other samples was heterozygous for the C and T alleles (black squares); HeLa genomic DNA (b,c) showed a 2:1 allelic ratio (CCT) reflecting HeLa's triploid 5q karyotypic status. In e, cDNA and genomic DNA from lymphoblastoid cell lines from ten different heterozygous individuals were analyzed: the ten different colors represent the ten individuals; technical replicates from the same cell line are connected by line segments. HeLa, SNU182 and the lymphoblastoid cell lines expressed the C allele of rs10065172 more strongly than the T allele (b-e); HCT116 and primary bronchial cells expressed the T allele much more strongly than the C allele (f,g).


figf3 ( a ) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six - to eightfold in HeLa cells . Cells were transfected with control ( siControl ) or IRGM - targeted ( siIRGM1 , siIRGM2 ) siRNA duplexes and assayed 48 h later by quantitative RT - PCR . Error bars , s . d . ( b ) IRGM knockdown reduces the efficiency of anti - bacterial autophagy . HeLa cells stably expressing LC3 - GFP ( a marker for autophagic vesicles ) were transfected with control or IRGM - directed siRNA oligos , then infected with S . typhimurium after 48 h . The percentage of bacteria encapsulated in LC3 - positive vesicles was determined by microscopy . Reductions in IRGM expression resulted in a significant loss of anti - bacterial autophagy compared to control cells . ( c ) Microscopic examination reveals a decreased number of autophagically encapsulated bacteria in IRGM knockdown cells . Control siRNA - treated ( siControl ) and IRGM siRNA - treated ( siIRGM1 , siIRGM2 ) cells were infected with S . typhimurium SL1344 and imaged by confocal microscopy . Control cells show numerous bacteria ( red in merged image ) surrounded by LC3 - GFP membranes ( green in merged image ) . Such bacteria - containing autophagosomes ( indicated by dashed boxes ) were almost completely absent in IRGM - deficient cells . The actin cytoskeleton was visualized using phalloidin ( blue in merge ) . Images are flat projections of confocal z - stacks . Scale bars , 10 μm . ( ( d ) HeLa cells stably expressing LC3 - GFP were transfected with increasing amounts of an IRGM expression construct and infected 24 h later with S . typhimurium . The percentage of bacteria found within LC3 - positive vesicles increased with increasing amounts of exogenous IRGM . Protein expression levels were confirmed by blotting lysates for Flag - IRGM expression , using an antibody to Flag to detect exogenous IRGM and actin as a loading control ( lower panel ) . Error bars in b and d , s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six- to eightfold in HeLa cells. Cells were transfected with control (siControl) or IRGM-targeted (siIRGM1, siIRGM2) siRNA duplexes and assayed 48 h later by quantitative RT-PCR. Error bars, s.d.(b) IRGM knockdown reduces the efficiency of anti-bacterial autophagy. HeLa cells stably expressing LC3-GFP (a marker for autophagic vesicles) were transfected with control or IRGM-directed siRNA oligos, then infected with S. typhimurium after 48 h. The percentage of bacteria encapsulated in LC3-positive vesicles was determined by microscopy. Reductions in IRGM expression resulted in a significant loss of anti-bacterial autophagy compared to control cells.(c) Microscopic examination reveals a decreased number of autophagically encapsulated bacteria in IRGM knockdown cells. Control siRNA-treated (siControl) and IRGM siRNA-treated (siIRGM1, siIRGM2) cells were infected with S. typhimurium SL1344 and imaged by confocal microscopy. Control cells show numerous bacteria (red in merged image) surrounded by LC3-GFP membranes (green in merged image). Such bacteria-containing autophagosomes (indicated by dashed boxes) were almost completely absent in IRGM-deficient cells. The actin cytoskeleton was visualized using phalloidin (blue in merge). Images are flat projections of confocal z-stacks. Scale bars, 10 μm. ((d) HeLa cells stably expressing LC3-GFP were transfected with increasing amounts of an IRGM expression construct and infected 24 h later with S. typhimurium. The percentage of bacteria found within LC3-positive vesicles increased with increasing amounts of exogenous IRGM. Protein expression levels were confirmed by blotting lysates for Flag-IRGM expression, using an antibody to Flag to detect exogenous IRGM and actin as a loading control (lower panel). Error bars in b and d, s.e.m.


Figure 1Localization of R - LPS ( green ) and S - LPS ( red ) in exponential phase bacteria . Scale bars : 2 μm . Distribution of R - LPS and S - LPS in exponential phase culture analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) . Numbers in each corner corresponds to % of relative frequencies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Localization of R-LPS (green) and S-LPS (red) in exponential phase bacteria. Scale bars: 2 μm. Distribution of R-LPS and S-LPS in exponential phase culture analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Numbers in each corner corresponds to % of relative frequencies.


Figure 2Roughness measurements on R - LPS labeled ( green ) WT and Δgmd cells . AFM images of whole bacteria and of the separated areas ( coloured squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The arithmetic roughness Ra is indicated below each area . Scale bars : 1 μm . Quantitative roughness measurement of R - LPS labeled WT and Δgmd cells . The areas with the lower roughness ( more regular surfaces ) of Δgmd cells were assigned as areas 1 . mAb : monoclonal antibody . Ab : antibody . nWT = 13 bacteria . nΔgmd = 7 bacteria . Differences were statistically analyzed by t - test . * * P < 0 . 01 . ns : not significant . Detection of forces between AFM tip functionalized with mAb against R - LPS and unlabeled WT cells . Height image and the corresponding adhesion image are shown on top and zooms of depicted areas ( black squares , 0 . 4 x 0 . 4 μm2 ) below . The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area . Scale bar : 1 μm . Correlation between roughness and detection of R - LPS on unlabeled WT cells . The areas with the lower roughness ( more regular surfaces ) were assigned as areas 1 . The boxplot represents the mean values ( squares ) , the median ( horizontal line ) , the 25 % and 75 % quartiles ( box limits ) and the standard deviations ( whiskers ) . n = 9 bacteria . Error bars : s . d . Statistical differences were analyzed by t - test . * * P < 0 . 01 . * P < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Roughness measurements on R-LPS labeled (green) WT and Δgmd cells. AFM images of whole bacteria and of the separated areas (coloured squares in first images, 0.4 x 0.4 μm2) are shown. The arithmetic roughness Ra is indicated below each area. Scale bars: 1 μm. Quantitative roughness measurement of R-LPS labeled WT and Δgmd cells. The areas with the lower roughness (more regular surfaces) of Δgmd cells were assigned as areas 1. mAb: monoclonal antibody. Ab: antibody. nWT = 13 bacteria. nΔgmd = 7 bacteria. Differences were statistically analyzed by t-test. **P < 0.01. ns: not significant.Detection of forces between AFM tip functionalized with mAb against R-LPS and unlabeled WT cells. Height image and the corresponding adhesion image are shown on top and zooms of depicted areas (black squares, 0.4 x 0.4 μm2) below. The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area. Scale bar: 1 μm. Correlation between roughness and detection of R-LPS on unlabeled WT cells. The areas with the lower roughness (more regular surfaces) were assigned as areas 1. The boxplot represents the mean values (squares), the median (horizontal line), the 25% and 75% quartiles (box limits) and the standard deviations (whiskers). n = 9 bacteria. Error bars: s.d. Statistical differences were analyzed by t-test. **P < 0.01. *P < 0.05.


Figure 3Co - localization of Omp2b ( red ) and R - LPS ( green ) in exponential phase bacteria . Scale bars : 2 μm . Co - detection of Omp2b ( red ) and R - LPS by AFM . R - LPS was detected using AFM tips functionalized with mAb against R - LPS . AFM images of whole bacteria and of the separated areas ( red squares in first images , 0 . 4 x 0 . 4 μm2 ) are shown . The percentages of adhesion Padh are indicated below each area . Scale bars : 1 μm . Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area . Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure . nWT = 13 bacteria , nΔgmd = 13 bacteria . Statistical analysis with Mann - Whitney U test showed * * P < 0 . 01 .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm.Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm.Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area. Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure. nWT = 13 bacteria, nΔgmd = 13 bacteria. Statistical analysis with Mann-Whitney U test showed **P < 0.01.


Figure 4Localization of initially labeled Omp25 ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . As a control , Omp25 localization after pulse - chase labeling with TRSE is shown at the bottom panel . Scale bars : 2 μm . Localization of initially labeled Omp2b ( green ) on TRSE - labeled ( red ) bacteria before ( 0 h ) and after 2 h of growth in the absence of both labelings ( 2 h ) . Scale bars : 2 μm . Co - localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels , respectively . Error bars correspond to s . d . from independent experiments . Differences were statistically analyzed by t - test . * * * P < 0 . 001 . nOmp25 = 279 bacteria , 85666 pixels ( 5 biological replicates ) . nOmp2b = 252 bacteria , 74630 pixels ( 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm.Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm.Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).


Figure 5Short pulse labeling of exponential phase bacteria with the fluorescent D - amino acid HADA . Merge is showing old pole marker PdhS - mCherry ( red ) and PG insertion sites ( white ) . Scale bars : 2 μm . Demographic representation of HADA labeling . Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS - mCherry fluorescence signal . NFI : normalized fluorescence intensity . n = 393 bacteria ( 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm.Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates).


Figure 6Localization of S - LPS ( green ) in the eFluor - labeled ( magenta ) inducible rough strain Δgmd plac - gmd possessing the old pole marker PdhS - mCherry ( red ) . The strains Δgmd and Δgmd plac - gmd - IPTG were used as negative controls . Scale bars : 2 μm . Demograph represents fluorescence profile of S - LPS labeling from ( A ) . Cells were oriented with the old pole at the bottom of the graph by PdhS - mCherry and sorted by cell length . NFI : normalized fluorescence intensity . n = 468 bacteria ( 3 biological replicates ) . Short pulse labeling of LPS ( 2 h ) by Kdo - N3 ( green ) on eFluor - labeled ( magenta ) bacteria expressing the old pole marker PdhS - mCherry ( red ) . Scale bars : 2 μm . Demographs represent fluorescence intensities of eFluor ( OM , left ) and Kdo - N3 ( LPS , right ) labeling . Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS - mCherry localization . NFI : normalized fluorescence intensity . n = 566 bacteria ( 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm.Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates).Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm.Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).


Figure 7Distribution of R - LPS and S - LPS in Δgmd plac - gmd after 3 , 6 , 9 and 24 h post - induction analyzed by flow cytometry ( one representative example among 3 biological replicates , n = 20 , 000 events ) . Quantification of S - LPS labeling from ( A ) as relative frequencies . Error bars correspond to s . d . from 3 independent experiments . n0h = 77 bacteria , n3h = 254 bacteria , n6h = 616 bacteria , n9h = 937 bacteria , n24h = 1059 bacteria .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Distribution of R-LPS and S-LPS in Δgmd plac-gmd after 3, 6, 9 and 24 h post-induction analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events).Quantification of S-LPS labeling from (A) as relative frequencies. Error bars correspond to s.d. from 3 independent experiments. n0h = 77 bacteria, n3h = 254 bacteria, n6h = 616 bacteria, n9h = 937 bacteria, n24h = 1059 bacteria.


Figure 3Typical retinal flat mount from Acta1GFP / + : Acta1 + / + bone marrow chimeras 20 weeks after reconstitution . Donor - derived GFP + Iba1 + cells ( arrow ) and GFP - Iba1 + resident microglia ( asterisks ) are shown . Pictures are representative for three animals from one experiment . Scale bars represents 50 µm . Microscopy - based quantification of GFP + Iba1 + retinal microglia in the inner ( IPL ) and outer plexiform layer ( OPL ) . One symbol represents one mouse . Data are presented as mean ± s . e . m . Direct fluorescence microscopic visualization for YFP ( green ) , the macrophage marker Iba1 ( red ) and DAPI for the nuclei ( blue ) at P0 . YFP + Iba1 + double - positive cells are marked by arrows . YFP - Iba1 + single - positive cells are labelled by asterisks . Representative images out of five examined animals are shown . Scale bars represents 25 µm . Quantitative analysis of regional YFP expression in Iba1 + macrophages in TAM - induced and untreated Cx3cr1CreERT2 : Rosa26 - YFP mice . Bars represent means ± s . e . m . Quantification was done from three ( untreated ) or five ( TAM ) mice obtained from one ( untreated ) or two ( TAM ) independent experiments . Level of significance determined by Mann - Whitney test between TAM and untreated revealed * p < 0 . 05 and Kruskal - Wallis test between retina , ciliary body and cornea revealed * p = 0 . 0204 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Typical retinal flat mount from Acta1GFP/+:Acta1+/+ bone marrow chimeras 20 weeks after reconstitution. Donor-derived GFP+Iba1+ cells (arrow) and GFP-Iba1+ resident microglia (asterisks) are shown. Pictures are representative for three animals from one experiment. Scale bars represents 50 µm.   Microscopy-based quantification of GFP+Iba1+ retinal microglia in the inner (IPL) and outer plexiform layer (OPL). One symbol represents one mouse. Data are presented as mean ± s.e.m.  Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP+Iba1+ double-positive cells are marked by arrows. YFP-Iba1+ single-positive cells are labelled by asterisks. Representative images out of five examined animals are shown. Scale bars represents 25 µm.   Quantitative analysis of regional YFP expression in Iba1+ macrophages in TAM-induced and untreated Cx3cr1CreERT2:Rosa26-YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann-Whitney test between TAM and untreated revealed *p < 0.05 and Kruskal-Wallis test between retina, ciliary body and cornea revealed *p = 0.0204.


Figure 6Above : Representative funduscopic pictures from living healthy Cx3cr1CreERT2 : Rosa26 - tdTomato mice on d0 . Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato + microglia before the laser - induced lesion formation . Below : Corresponding immunofluorescence pictures . Non - lesioned retina show a regular pattern of Iba1 + ( green ) tomato + ( red ) retinal microglia while macrophages are absent on the retinal pigment epithelium ( RPE ) under native conditions . Pictures are representative for six mice analyzed in one experiment . Scale bars represents 50 µm . c ) Above : In vivo funduscopy on d7 post lesion . Funduscopic and red fluorescence image depict the lesions ( encircled with dashed white lines ) and accumulation of tomato + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization . Below : Representative immunofluorescence for Iba1 ( green ) in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Resident retinal microglia are Iba1 + tomato + ( asterisks ) whereas blood - derived myeloid cells are Iba1 + tomato - ( arrows ) and accumulate at sites of laser - induced CNV . Overlay is shown left . Typical pictures from six mice obtained from one independent experiment are shown . Scale bars represents 50 µm . d ) Percentage ( left ) and absolute numbers ( right ) of myeloid subsets in the retina at different time points post lesion . Red columns , red lines and red symbols represent tomato * Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice whereas green columns , green lines and green symbols represent blood - derived tomato - Iba1 + myeloid cells . Left , Wilcoxon test at d3 ( ns p = 0 . 0625 ) , d28 ( ns , p = 0 . 25 ) and d56 ( ns p = 0 . 125 ) and paired t test at d7 ( * * * p < 0 . 0001 and d14 ( * * * p < 0 . 0001 ; right , absolute numbers Kruskal - Wallis test ( tomato * Iba1 + * p < 0 . 05 , tomato - Iba1 + * * p < 0 . 01 ) . Data represent means ± s . e . m . from at least three mice per group ( two to six lesion per mouse ) out of one ( d0 , d14 , d28 , d56 ) or two ( d7 , d14 ) independent experiments . e ) Distribution ( left ) and absolute numbers ( right ) of myeloid cells in the RPE at different time points following laser - induced lesion . Red columns , red lines and red symbols represent Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Green columns , green lines and green symbols represent blood - derived Iba1 + tomato - myeloid cells . Left , paired t test at d3 , d7 , d14 ( * * p < 0 . 01 , * * * p < 0 . 001 ) , Wilcoxon test at d28 and d56 ( ns p > 0 . 05 ) ; right , absolute numbers Kruskal - Wallis test ( ns p > 0 . 05 ) . Data represent means ± s . e . m . from at least three mice per group ( two to six lesion per mouse ) out of one ( d0 , d14 , d28 , d56 ) or two ( d7 , d14 ) independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Above: Representative funduscopic pictures from living healthy Cx3cr1CreERT2:Rosa26-tdTomato mice on d0. Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato+ microglia before the laser-induced lesion formation. Below: Corresponding immunofluorescence pictures. Non-lesioned retina show a regular pattern of Iba1+ (green) tomato+ (red) retinal microglia while macrophages are absent on the retinal pigment epithelium (RPE) under native conditions. Pictures are representative for six mice analyzed in one experiment. Scale bars represents 50 µm. c) Above: In vivo funduscopy on d7 post lesion. Funduscopic and red fluorescence image depict the lesions (encircled with dashed white lines) and accumulation of tomato+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice. Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization. Below: Representative immunofluorescence for Iba1 (green) in Cx3cr1CreERT2:Rosa26-tdTomato mice. Resident retinal microglia are Iba1+tomato+ (asterisks) whereas blood-derived myeloid cells are Iba1+tomato- (arrows) and accumulate at sites of laser-induced CNV. Overlay is shown left. Typical pictures from six mice obtained from one independent experiment are shown. Scale bars represents 50 µm.d) Percentage (left) and absolute numbers (right) of myeloid subsets in the retina at different time points post lesion. Red columns, red lines and red symbols represent tomato*Iba1+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice whereas green columns, green lines and green symbols represent blood-derived tomato-Iba1+ myeloid cells. Left, Wilcoxon test at d3 (ns p = 0.0625), d28 (ns, p = 0.25) and d56 (ns p = 0.125) and paired t test at d7 (***p < 0.0001 and d14 (***p < 0.0001; right, absolute numbers Kruskal-Wallis test (tomato*Iba1+ *p < 0.05, tomato-Iba1+ ** p < 0.01). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments. e) Distribution (left) and absolute numbers (right) of myeloid cells in the RPE at different time points following laser-induced lesion. Red columns, red lines and red symbols represent Iba1+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice. Green columns, green lines and green symbols represent blood-derived Iba1+tomato- myeloid cells. Left, paired t test at d3, d7, d14 (**p < 0.01, ***p < 0.001), Wilcoxon test at d28 and d56 (ns p > 0.05); right, absolute numbers Kruskal-Wallis test (ns p > 0.05). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments.


Figure 9a ) H2 - Aa expression visualized by trajectory analysis and represented in a t - SNE plot . b ) H2 - Aa immunohistochemistry ( green ) by Iba1 + ( red ) microglia inside lesioned and non - lesioned areas in the retina and RPE . Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative . Picture is representative of two mice . In the RPE , arrow heads label Tom - MHCII + cells . Scale bars represents 25 µm . c ) HLA - DRA mRNA expression examined by RNA - Seq via Massive analysis of cDNA ends ( MACE ) analysis of human formalin - fixed and paraffin - embedded ( FFPE ) membranes of choroidal neovascularization ( CNV ) . Bars represent means ± s . e . m . of four investigated human samples of age - related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE . Level of significance was calculated using DESeq ( p = 3 . 61x10 - 21 ) . d ) Immunohistochemistry for HLA - DR in the RPE / choroid of one control and the CNV membrane of one patient suffering from age - related macular degeneration associated with choroidal neovascularization .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9a) H2-Aa expression visualized by trajectory analysis and represented in a t-SNE plot.b) H2-Aa immunohistochemistry (green) by Iba1+ (red) microglia inside lesioned and non-lesioned areas in the retina and RPE. Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative. Picture is representative of two mice. In the RPE, arrow heads label Tom-MHCII+ cells. Scale bars represents 25 µm.c) HLA-DRA mRNA expression examined by RNA-Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin-fixed and paraffin-embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age-related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (p = 3.61x10-21).d) Immunohistochemistry for HLA-DR in the RPE/choroid of one control and the CNV membrane of one patient suffering from age-related macular degeneration associated with choroidal neovascularization.


Figure 1 ( A ) Levels of Piwil1 ( Miwi ) , Piwil2 ( Mili ) and Piwil4 ( Miwi2 ) transcripts in RNA seq . data from undifferentiated aNPCs ( Days of differentiation - DIF - 0 ) and differentiating neuroblasts ( DIF 4 - 14 ) ; insets in left and right panels show the same data with smaller scales in the ordinate axes . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . ( B ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral - induced neurogenesis ( DIF4 - 14 ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test ( C , D ) Western blot ( insets ) and quantification ( bar graphs ) of Miwi and Mili protein abundance in lysates from postnatal mouse testis , hippocampus and undifferentiated aNPCs cultures . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test ( E ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by two - tailed Student ' s t - test ( in E ) . ( F ) Representative immunofluorescence micrograph of Mili ( white ) and Td + NSCs ( red ) in hippocampal subgranular zone ( SGZ ) ; arrows indicate Td + Mili + double - positive cells . Data information : In F : GCL , granular cell layer ; H , Hilus . The scale bars represent 10 µm . ( G ) Scheme of the experiment ( top ) and Mili mRNA expression in sorted Td + and Td - cells after in vivo transduction with split - Cre viruses in the hippocampus ( bottom ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test G )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Levels of Piwil1 (Miwi), Piwil2 (Mili) and Piwil4 (Miwi2) transcripts in RNA seq. data from undifferentiated aNPCs (Days of differentiation -DIF- 0) and differentiating neuroblasts (DIF 4-14); insets in left and right panels show the same data with smaller scales in the ordinate axes. Data information: data are expressed as mean ± SEM, n = 3 biological replicates.(B) Western blot (inset) and quantification (bar graph) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral-induced neurogenesis (DIF4-14). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test(C, D) Western blot (insets) and quantification (bar graphs) of Miwi and Mili protein abundance in lysates from postnatal mouse testis, hippocampus and undifferentiated aNPCs cultures. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test(E) Western blot (inset) and quantification (bar graph) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by two-tailed Student's t-test (in E).(F) Representative immunofluorescence micrograph of Mili (white) and Td+ NSCs (red) in hippocampal subgranular zone (SGZ); arrows indicate Td+ Mili + double-positive cells. Data information: In F: GCL, granular cell layer; H, Hilus. The scale bars represent 10 µm.(G) Scheme of the experiment (top) and Mili mRNA expression in sorted Td+ and Td- cells after in vivo transduction with split-Cre viruses in the hippocampus (bottom). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test G)


Figure 2 ( E ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA ( Control ) or shRNA targeting Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM unless differently indicated , n = 3 biological replicates ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . Western blot ( G ) of the endogenous piCS after co - immunoprecipitation ( IP ) with endogenous Mili ( IP Mili ) , or control IgG ( IP IgG ) in lysates of DIF4 aNPCs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(E) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA (Control) or shRNA targeting Mili (Mili KD). Data information: data are expressed as mean ± SEM unless differently indicated, n = 3 biological replicates ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.Western blot (G) of the endogenous piCS after co-immunoprecipitation (IP) with endogenous Mili (IP Mili), or control IgG (IP IgG) in lysates of DIF4 aNPCs.


Figure 4 ( C ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 ( C , biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . ( E ) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled ( Control ) and GapmeR1 against Mili ( Mili KD ) ; Right panels : Fold change in GFAP fluorescence intensity level ( upper graph ) in a hippocampal region of interest ( ROI ) of 500µm2 in brain slices upon Mili KD compared to Control ; Gfap mRNA levels ( lower graph ) in the DG from mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 E mRNA and 5 ( E biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . SGZ , subgranular zone . The scale bars represent 100 µm ( E , ( F ) ( Left ) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP ( green ) , BrdU ( red ) , NeuN ( white ) and nuclear DNA ( blue ) at 30 dpi of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) ; ( right graphs ) percentages of NeuN + BrdU + ( white arrowheads in the images ) , or GFAP + BrdU + ( yellow arrowheads in the images ) double - positive cells over total BrdU + cells . Data information : data are expressed as mean ± SEM , n = F ) biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . GCL , granular cell layer , SGZ , subgranular zone . The scale bars represent 100 µm F ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 against Mili (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 (C, biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.(E) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled (Control) and GapmeR1 against Mili (Mili KD); Right panels: Fold change in GFAP fluorescence intensity level (upper graph) in a hippocampal region of interest (ROI) of 500µm2 in brain slices upon Mili KD compared to Control; Gfap mRNA levels (lower graph) in the DG from mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 E mRNA and 5 (E biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. SGZ, subgranular zone. The scale bars represent 100 µm (E,(F) (Left) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP (green), BrdU (red), NeuN (white) and nuclear DNA (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili (Mili KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells. Data information: data are expressed as mean ± SEM, n = F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 100 µm F).


Figure 5 ( B ) Representative bright - field microscopy images ( left ) and quantification ( right ) of ß - galactosidase + aNPCs as percent of total cells upon Mili KD , or control 48 h after induction of spontaneous differentiation . Data information : data are expressed as mean ± SEM , n = 5 ( B biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm ( B , ( C ) Representative fluorescence microscopy images ( left ) and quantification ( right ) of control or Mili KD neuroblasts 48h after spontaneous differentiation , immunostained with anti - BrdU ( white ) and Ki67 ( purple ) antibodies . ( Right ) Percentage of BrdU + and Ki67 - cells over BrdU + cells . Data information : data are expressed as mean ± SEM , n = C ) biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm C )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Representative bright-field microscopy images (left) and quantification (right) of ß-galactosidase+ aNPCs as percent of total cells upon Mili KD, or control 48 h after induction of spontaneous differentiation. Data information: data are expressed as mean ± SEM, n = 5 (B biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm (B,(C) Representative fluorescence microscopy images (left) and quantification (right) of control or Mili KD neuroblasts 48h after spontaneous differentiation, immunostained with anti-BrdU (white) and Ki67 (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells. Data information: data are expressed as mean ± SEM, n = C) biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm C)


Figure 1Kainic acid mouse model of TLE : Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV - pDyn delivery ( 2x109 gp ) . higher time resolution of the indicated section of the Day + 1 in ( B ) , representing a hippocampal paroxysmal discharge ( HPD ) . A generalized seizure is depicted in Fig . EV1 D and E : The characteristic EEG features of this model , secondary generalized seizures ( bars ) and HPDs ( lines ) were reduced in number and in duration by AAV - pDyn ( red ; n = 3 ( from day 60 ) ; 7 ( till day 30 ) per time - interval ) , but not by AAV - ΔGFP or after sham treatment ( blue ; n = 4 ( day 90 ) ; 5 ( day 30 and 60 ) ; 6 ( before day 30 ) , per time - interval ) . The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE . Injection of norBNI ( 20 mg / kg ; i . p . ) results in a transient reappearance of HPDs immediately and 24 hrs after application . One week after norBNI application ( washout ) suppression of HPDs was re - established . Data obtained from 4 epileptic animals before ( black ) and after ( red ) AAV - pDyn delivery ( 2x109 gp ) are depicted . * EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self - sustained status epilepticus ( SSSE ) before ( black ) and after ( red ) AAV - pDyn delivery ( 4x109 gp ) are depicted . Spike trains with a frequency of at least 2 . 5 Hz induced by SSSE were markedly reduced by AAV - pDyn ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2x109 gp).higher time resolution of the indicated section of the Day +1 in (B), representing a hippocampal paroxysmal discharge (HPD).A generalized seizure is depicted in Fig. EV1 D and E: The characteristic EEG features of this model, secondary generalized seizures (bars) and HPDs (lines) were reduced in number and in duration by AAV-pDyn (red; n = 3 (from day 60); 7 (till day 30) per time-interval), but not by AAV-ΔGFP or after sham treatment (blue; n = 4 (day 90); 5 (day 30 and 60); 6 (before day 30), per time-interval). The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE.Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 hrs after application. One week after norBNI application (washout) suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2x109 gp) are depicted. *EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self-sustained status epilepticus (SSSE) before (black) and after (red) AAV-pDyn delivery (4x109 gp) are depicted. Spike trains with a frequency of at least 2.5 Hz induced by SSSE were markedly reduced by AAV-pDyn (n=4).