Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 2. Yap and Taz levels and YAP/TAZ downstream targets are elevated in the forebrains of Tsc2 cKO mice. (A) Western blots for E16.5 WT (n = 5) and Tsc2 cKO mice (n = 5). (B) Quantification of protein levels using densitometry shows significant reductions in Tsc2 but not pYap levels and significant increases in Yap/Taz levels. Data are presented as the mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by unpaired, two-tailed t-test. Tsc2 (t = 9.889, df = 8, P ≤0.0001), Yap (t = 3.905, df = 8, P = 0.0045), pYap/Yap (t = 1.572, df = 8, P = 0.1546), Taz (t = 2.684, df = 8, P = 0.0278). (C) Immunostaining shows that Yap/Taz levels are increased in E16.5 Tsc2 cKO animals. Scale = 100 μm, inset =25 μm. (D) Representative image of an RNAscope ® in situ hybridization assay with probe for Ccn2 mRNA in P12 WT (n = 3) and Tsc2 cKO (n = 3). Tsc2 cKO mice show elevations in Ccn2 in the corpus callosum. Scale = 100 μm.

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Western Blot, Two Tailed Test, Immunostaining, RNAscope, In Situ Hybridization

Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 3. Yap/Taz/Tsc2 tcKO or verteporfin treatment reduces corti- cal thickness increases seen in Tsc2 cKO mice. (A) Cortical thick- ness increases are detectable in Tsc2 cKO animals and rescued in tcKO animals at E16.5. Scale bar = 100 μm. WT n = 6; Tsc2 cKO n = 5; Yap/Taz/Tsc2 tcKO n = 3. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by one-way ANOVA (ANOVA, F = 16.38, df = 2,11, P = 0.0005; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO = 0.0006, Adjusted PWT-tcko = 0.8958, Adjusted PTSC2 cKO-tcKO = 0.0048). (B) Cortical thickness increases are also rescued in tcKO animals when compared with Tsc2 cKO animals at P0. Scale bar = 100 μm. WT n = 11; Tsc2 cKO n = 10; Yap/Taz/Tsc2 tcKO n = 7. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by one-way ANOVA (ANOVA, F = 14.66, df = 2,25, P ≤0.0001; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO ≤0.001, Adjusted PWT-tcko = 0.01919, Adjusted PTSC2 cKO-tcKO = 0.0156). (C) Schematic for verteporfin or vehicle control treatment and collection experiment. (D) Cortical thickness of Tsc2 cKO mice is decreased following 5 days of intraperitoneal verteporfin treatment at P0 compared with DMSO only treatment. Scale = 100 μm. WT + DMSO n = 4; Tsc2 cKO + DMSO n = 5; Tsc2 cKO + Verteporfin n = 5. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by one- way ANOVA (ANOVA, F = 38.93, df = 2,11, P ≤0.0001; Tukey’s multiple comparisons test, Adjusted PWT + DMSO-TSC2 cKO + DMSO ≤0.0001, Adjusted PWT + DMSO-TSC2 cKO + Vert = 0.0005, Adjusted PTSC2 cKO + DMSO-TSC2 cKO + Vertt

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Control

Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 4. tcKO of Yap/Taz/Tsc2 rescues cortical thickness increases caused by Tsc2 cKO through a reduction in cell size. (A) Cortical cell size is increased in Tsc2 cKO but reduced to WT levels in tcKO at E16.5. Scale bar = 100 μm, 25 μm for high magnification images. (B) For E16.5, WT n = 4 animals (400 cells); Tsc2 cKO n = 4 animals (400 cells); Yap/Taz/Tsc2 tcKO n = 3 animals (300 cells). Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a one-way ANOVA (ANOVA, F = 13.63, df = 21 097, P ≤0.0001; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO ≤0.0001, Adjusted PWT-tcko = 0.0940, Adjusted PTSC2 cKO-tcKO = 0.0172). (C) Soma size of late-born neurons is increased in the lower bins of Tsc2 cKO animals, which is reversed in tcKO animals at P0. Scale bar = 100 μm. (D) For P0, WT n = 8 animals (800 cells); Tsc2 cKO n = 8 animals (800 cells); Yap/Taz/Tsc2 tcKO n = 7 animals (700 cells). Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a one-way ANOVA (ANOVA, F = 17.03, df2,2295=, P ≤0.0001; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO ≤0.0001, Adjusted PWT-tcko = 0.0074, Adjusted PTSC2 cKO-tcKO = 0.0239). (E) Soma size of late-born neurons is increased in the upper bins of Tsc2 cKO animals, which is reversed in tcKO animals at P12. Scale bar = 100 μm. (F) For P12, WT n = 4 animals (200 cells top 5 bins); Tsc2 cKO n = 3 animals (150 cells top 5 bins); Yap/Taz/Tsc2 tcKO n = 3 animals (150 cells top 5 bins). Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a one-way ANOVA (ANOVA, F=, df = 2876, P ≤0.0001; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO = 0.0003, Adjusted PWT-tcko = 0.9773, Adjusted PTSC2 cKO-tcKO = 0.0008).

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques:

Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 5. tcKO of Yap/Taz/Tsc2 rescues late-born neuromigration deficits in the cortex when compared with Tsc2 cKO mice. (A) Immunostaining for Cux1, a late-born neuron marker, shows that a greater proportion of late-born neurons that fail to reach the top of the cortical plate in Tsc2 cKO animals when compared with WT and tcKO mice. Scale bar = 100 μm. (B) Quantification of late-born neuron migration in P0 mice. WT n = 9; Tsc2 cKO n = 9; tcKO n = 6. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a two-way ANOVA (ANOVA, FInteraction = 2.445, FBin = 52.12, FGroup = 5.502e-006, dfInteraction = 18 210, dfBin = 9210, dfGroup = 2210, PInteraction = 0.004, PBin ≤0.001, PGroup ≥0.99; Tukey’s multiple comparisons test, Adjusted PWT-tcKO, Bin 10 = 0.05, Adjusted PWT-TSC2,Bin 8 ≤0.001, Adjusted PWT-tcKO,Bin 8 = 0.010). (C) Late-born neurons, as visualized by Cux1 staining, remain ‘stuck’ in the lower area of the cortex at P12. Scale bar = 100 μm. (D) Quantification of late-born neuron migration in P12 mice. WT n = 6; Tsc2 cKO n = 4; tcKO n = 5. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a one-way ANOVA (ANOVA, FInteraction = 8.100, FBin = 57.19, FGroup = 1.991e −014, dfInteraction = 18 110, dfBin = 9110, dfGroup = 2110, PInteraction ≤0.001, PBin ≤0.001, PGroup ≥0.99; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO, Bin 10 ≤0.001, Adjusted PWT-tcKO, Bin 10 = 0.004, Adjusted PTSC2 cKO-tcKO, Bin 10 = 0.03, Adjusted PWT-TSC2 cKO, Bin 9 ≤0.001, Adjusted PTSC2 cKO-tcKO, Bin 9 ≤0.001, Adjusted PWT-tcKO, Bin 7 ≤0.001, Adjusted PTSC2 cKO-tcKO, Bin 7 ≤0.001, Adjusted PWT-tcKO, Bin 5 = 0.04, Adjusted PWT-tcKO, Bin 5 = 0.04, Adjusted PWT-TSC2 cKO, Bin 5 = 0.007).

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Immunostaining, Marker, Migration, Staining

Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 6. tcKO of Yap/Taz/Tsc2 rescues neuromigration deficits found in the hippocampus when compared with Tsc2 cKO mice. (A) H&E staining and Ctip2 immunostaining demonstrate a split hippocampus phenotype in Tsc2 cKO animals. tcKO animals show an improvement in their hippocampal phenotype, although some ectopic cells can be found. Scale bar = 100 μm. (B) Quantification of the distribution of Ctip2-positive cells within a measured area of the CA1 region. WT n = 8; Tsc2 cKO n = 5; Yap/Taz/Tsc2 tcKO n = 5. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a two-way ANOVA (ANOVA, FInteraction = 5.342, FBin = 47.19, FGroup = 3.207e-012, dfInteraction = 18 150, dfBin = 9150, dfGroup = 2150, PInteraction ≤0.0001, PBin ≤0.0001, PGroup ≥0.9999; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO, Bin 7 = 0.0002, Adjusted PTSC2 cKO-tcKO, Bin 7 = 0.0117, Adjusted PWT-TSC2 cKO, Bin 6 = 0.0496). (C) Individual quantification of the distribution of Ctip2-positive cells in the two significantly different binned areas. WT n = 8; Tsc2 cKO n = 5; Yap/Taz/Tsc2 tcKO n = 5. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following the two-way ANOVA described in B. See B for statistics. (D) Immunostaining for Ctip2 in P12-P15 mice shows that Tsc2 cKO animals show the presence of distinct rings of neurons within the stratum lacunosum moleculare layer. Some tcKO animals present with rings, but this is not found in every animal. Scale bar = 100 μm. (E) Quantification of the number of rings found in each group of P12 mice demonstrates that the number of rings in tcKO animals is significantly reduced when compared with Tsc2 cKO animals. WT n = 9; Tsc2 cKO n = 7; Yap/Taz/Tsc2 tcKO n = 8. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001 by post-hoc Tukey test following a one-way ANOVA (ANOVA, F = 12.41, df = 2,21, P = 0.0003; Tukey’s multiple comparisons test, Adjusted PWT-TSC2 cKO = 0.0003, Adjusted PWT-tcko = 0.6679, Adjusted PTSC2 cKO-tcKO = 0.0.0027).

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Staining, Immunostaining

Journal: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 7. Myelination defects that are seen in Tsc2 cKO animals are improved in tcKO animals. (A) Immunostaining for Mbp in P12 animals shows alterations in myelin distribution in Tsc2 cKO and tcKO animals. Scale bar = 100 μm. (B) Quantification of the distribution of average intensity of Mbp staining within the Otsu-thresholded images of P12 animals. WT n = 5; Tsc2 cKO n = 5; Yap/Taz/Tsc2 tcKO n = 5. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.00005 by unpaired, two-tailed t-test. WT-Tsc2 cKO (t = 2.480, df = 7, P = 0.0422), WT-tcKO (t = 3.479, df = 8, P = 0.0083), cKO-tcKO (t = 0.2643, df = 7, P = 0.7992). (C) Immunostaining for Mbp in P15 animals shows alterations in myelin distribution in Tsc2 cKO and tcKO animals. Scale bar = 100 μm. (D) Quantification of the distribution of average intensity of Mbp staining within the Otsu-thresholded images of P15 animals. WT n = 4; Tsc2 n = 3; Yap/Taz/Tsc2 tcKO n = 3. Data are presented as mean ± SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.00005 by unpaired, two-tailed t-test. WT-Tsc2 cKO (t = 2.700, df = 5, P = 0.0428), WT-tcKO (t = 1.482, df = 5, P = 0.1984), cKO-tcKO (t = 4.663, df = 4, P = 0.0096).

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Immunostaining, Staining, Two Tailed Test

Journal: Science advances

Article Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex.

doi: 10.1126/sciadv.adr5807

Figure Lengend Snippet: Fig. 2. The TSC1 HR domain and the structural basis of WIPI3 binding. (A) Focused view of the TSC HR dimer. A single subunit of the TSC1 HR dimer mediates contacts to the TSC1 CC domain and the TSC2 HRs. Two regions of the large extended loop (TSC1 IDR1) shield the exposed second binding site of the TSC1 N-terminal dimer. Top: Schematic of the TSC1 HR dimer in context of TSC. (B) The TSC1 HR dimer clamps onto a hydrophobic helix of the TSC1 CC domain. The second TSC1 helix is hidden for clarity [shown in (A), dark blue]. (C) Key contacts in the TSC1 HR dimer interface mediated by the first TSC1 extended loop (IDR1). (D) Focused view of the WIPI3:TSC1 interaction as resolved by cryo-EM. (E) The 3.17-Å crystal structure of WIPI3 in complex with the TSC1 WIR motif. Bottom: Schematic of the WIPI3 binding site in context of TSC.

Article Snippet: TBC1D7 was polymerase chain reaction amplified from pET- 28a TBC1D7 (Addgene plasmid no. 32047 was a gift from C. Arrowsmith) and cloned into pcDNA 3.1 (Thermo Fisher Scientific) with a C- terminal FLAG- tag. pRK7 FLAG TSC2 (Addgene plasmid no. 8996) was a gift from J. Blenis, and pcDNA 3.1 myc TSC1 (Addgene plasmid no. 12133) was a gift from C. Walker.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science advances

Article Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex.

doi: 10.1126/sciadv.adr5807

Figure Lengend Snippet: Fig. 4. TSC:WIPI3 disease–associated mutations and model of the TSC:WIPI3:RHEB lysosomal mTORC1 inhibitory complex. (A) Cα atoms of all disease-associated missense mutations are rendered as spheres of size and color proportional to the frequency of observation (COSMIC, LOVD, and HGMD) (55–57). Disease-associated muta- tions cluster to the TSC1 HR PIP-binding dimer (left), as well as the TSC2 Rap-GAP central core (right). (B) Surface rendering of the complete human TSC with RHEB mod- elled according to AlphaFold (58). The binding sites of RHEB, TSC1:PI(3)P, and WIPI3:PI(3)P define a membrane binding plane that is consistent with a singly occupied RHEB-binding model of the TSC:WIPI3:RHEB lysosomal mTORC1 inhibitory complex.

Article Snippet: TBC1D7 was polymerase chain reaction amplified from pET- 28a TBC1D7 (Addgene plasmid no. 32047 was a gift from C. Arrowsmith) and cloned into pcDNA 3.1 (Thermo Fisher Scientific) with a C- terminal FLAG- tag. pRK7 FLAG TSC2 (Addgene plasmid no. 8996) was a gift from J. Blenis, and pcDNA 3.1 myc TSC1 (Addgene plasmid no. 12133) was a gift from C. Walker.

Techniques: Binding Assay, Membrane

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a , c ) Lung sections evaluated by H&E staining of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice at the age of ( a ) 3 and ( c ) 6 months. ( b ) Immunofluorescence for Mac-2 and p-S6 in lung sections of 3 month-old mice. Boxed region shows p-S6 staining only. ( d ) Image of a Tsc2 fl/fl Lyz2 -Cre mouse at the age of 6 months (left) and immunohistochemistry of Mac-2 of a paw (right). ( e ) Immunofluorescence for Mac-2 and F4/80 in paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice. ( f ) Flow cytometric scatterplot of the G1 population of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre peritoneal macrophages. ( g ) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice and evaluated after 3 months by histology. Left panel: H&E staining of lung sections. Right panel: IHC of Mac-2 in liver. ( h ) Flow cytometry analysis of hypertrophic macrophages in the lungs of Tsc2 fl/fl (n=5) and Tsc2 fl/fl Lyz2 -Cre (n= 4) mice. Shown are means. ( i ) Expression of the indicated mRNAs in the lungs of 6 month-old mice. Shown are means ± SE (n= 5). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant (Student's t test). Representative histological images or plots are from one mouse; each analysis was performed on three ( b , d , e , g ) or five ( a , c,f ) mice per genotype. Numerical data are representative of two independent ( h ) or cumulative of two independent ( i ) experiments. Scale bars, 100 μm ( a , c , e , g ), 10 μm ( b ), 1 mm ( d ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Injection, Irradiation, Flow Cytometry, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Immunoblots of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow-derived macrophages (BMDM), treated for 18 hours with 100 nM rapamycin or solvent control, hybridized with the indicated antibodies. ( b ) Surface expression of the indicated proteins on Tsc2 fl/fl (n=6) and Tsc2 fl/fl Lyz2 -Cre (n=4) BMDM after differentiation. ( c ) Left: Images of IL-4 stimulated (10 ng/ml) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM treated with solvent or 100 nM rapamycin for four days. Right: Quantification of cluster formation. Clusters in three pictures per condition (4x magnification) were counted (n= 3). ( d ) Flow cytometry scatterplot of CSF1-deprived BMDM on day 7. ( e,f ) Cell cycle analysis of ( e ) BMDM by 7-AAD and EdU staining and of ( f ) peritoneal macrophages by PI staining from Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice (n= 5). ( g ) IHC of Ki-67 in lung sections of 3 month-old Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice. ( h ) Proliferation analysis of BMDM stimulated with 40 ng/ml CSF1 and treated with solvent or 100 nM rapamycin (n=4). Shown are means ± SE ( b , f,h ) or SD ( c ). *p < 0.001 (Student's t test) ( c , f ). Data are representative of three ( a , d , g,c ) or two ( e ) or cumulative from two independent experiments ( b,f,h ) Scale bar, 100 µm ( c ), 50 μm ( g ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Western Blot, Derivative Assay, Solvent, Control, Expressing, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Scatterplot of global gene-expression profiles of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM. Blue dots, 426 genes significantly higher expressed in Tsc2 fl/fl BMDM; red dots, 401 genes significantly higher expressed in Tsc2 fl/fl Lyz2 -Cre BMDM. ( b ) Gene-set enrichment analysis (GSEA) of hallmark gene sets (H.all) from the Molecular Signatures Database of the Broad Institute, showing the most significantly enriched gene sets in Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM and their normalized enrichment scores (NES) as well as their false discovery rates (FDR). ( c,d ) GSEA plot of the E2F targets (c) and Tnfa signaling via NFKB (d) gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM from the analysis in (b) with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom). ( e ) GSEA of the 'KEGG_apoptosis' gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM. ( f ) The most significantly enriched transcription factor target gene sets (C3.TFT) from the Molecular Signatures Database in unstimulated Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM derived by GSEA. NES, normalized enrichment score; FDR, false discovery rate. Data are from one experiment with four biological replicates per genotype obtained from two independent experiments.

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Gene Expression, Derivative Assay

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a,b,c ) BMDM were treated with 100 nM rapamycin, solvent control and 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. ( d ) IHC of CDK4 in lung sections of 3 month-old mice. ( e ) Left, analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of the CKD4 inhibitor PD-0332991 (n= 4). Right, cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 µM PD-0332991 for 18 h (n= 3). ( f,g ) Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice. After ten days mice were treated daily with PD-0332991 (n=10) or solvent control (n=10) daily for two month. ( f ) Lung (left panel) and liver (right panel) sections were evaluated by IHC of Mac-2. ( g ) Area of Mac-2 positive cells compared to total lung area of the treated mice. Two random images per animal were evaluated. ( h ) BMDM were treated with 1 µM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SE ( e ) or SD ( g ). *p < 0.01, **p < 0.001 (Student's t test). Data are representative of three ( a , b , c , d ), one ( f,g ) or two ( h ) independent, or cumulative of two ( e ) experiments. Scale bar, 40 μm ( d ), 100 μm ( f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Western Blot, Cell Cycle Assay, Injection, Irradiation

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMDM reported in units of mpH/min and pmol/min, respectively (n=8). ( b ) Uptake of 2-NBDG in BMDM treated with solvent control or 100 nM rapamycin for 18 h; analyzed by flow cytometry (n=4). ( c ) Uptake of 2-NBDG in peritoneal macrophages from Tsc2 fl/fl (n=4) and Tsc2 fl/fl Lyz2 -Cre (n=5) mice. ( d ) Total amount of glucose in lung tissue of mice (n=10). ( e ) Mitotracker Green staining analyzed by flow cytometry of BMDM treated with solvent control or 100 nM rapamycin for 18 h (n=4). ( f ) Immunofluorescence for F-ATPase β of BMDM deprived of CSF1. ( g ) ECAR of Tsc2 fl/fl Lyz2 -Cre BMDM that were treated for 30 min with solvent or 1 µM PD-0332991 (n=8). ( h ) ECAR of Tsc2 fl/fl that were treated overnight with solvent or 10 ng/ml CSF1 and at the indicated time point with 1 µM PD-0332991 (n=8). ( i ) Images of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity on frozen lung sections of mice in situ . Sections were additionally stained for Mac-2, p-S6, and DAPI. ( j ) Fraction (in %) of p-S6-positive Mac-2 macrophages in lungs of mice with high activities of GAPDH, lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) (n=3-4). ( k ) Analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of 2-DG (n= 4). ( l ) Cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 mM 2-DG for 18 h (n= 3). ( m ) BMDM were treated with 1 mM 2-DG or solvent control and then stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SD ( a , g , h , j ) or SE ( b , c , e , k , l ) or boxplots with means, 25 th to 75 th percentile and minimum to maximum bars ( d ). *p<0.05, **p<0.001, ***p<0.001 (Student's t test). Data are representative of one ( d ) or two ( a,f-j,m ) independent experiments or cumulative of 2 experiments ( b,c,e,k,l) . Scale bar, 10 μm ( f ), 100 μm ( i ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Flow Cytometry, Staining, Immunofluorescence, Activity Assay, In Situ, Cell Cycle Assay, Western Blot

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Sections of three sarcoidosis patients stained with p-S6 by IHC. ( b ) Immunofluorescence for Mac-2 and p-S6 in a sarcoidosis granuloma. ( c ) GSEA analysis of the ‘mTORC1 signaling’, ‘E2F targets’, and ´Glycolysis´ gene signatures in progressive relative to self-limiting sarcoidosis from the data of Lockstone et al . ( d ) An unsupervised cluster analysis of the microarray data of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM performed with the genes that were differentially expressed in progressive relative to self-limiting sarcoidosis patients. ( e ) IHC for p-S6 and Ki-67 of granulomas from sarcoidosis patients. ( f ) Immunofluorescence for Mac-2 and Ki-67 in a sarcoidosis granuloma. ( g ) Relationship between p-S6 and Ki-67 expression in granulomas of 27 human sarcoidosis biopsies. Ki-67 high, > 5 % Ki-67 positive cells in the granuloma; Ki-67 low, < 5 % Ki-67 positive cells in the granulomas. The relationship was investigated using fisher’s exact test. Data are representative of 27 human sarcoidosis patients ( a , b , e , f ) or from 8 self-limiting and 7 progressive sarcoidosis patients ( c , d ). Scale bar, 200 μm ( a ), 20 μm ( b ), 100 μm ( e,f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Microarray, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) 2 month-old and ( b ) 6 month-old Tsc2 fl/fl Lyz2 -Cre mice were treated with placebo or everolimus for three weeks and lung sections were evaluated by H&E staining. ( c ) Paw and tail images of a 6 month-old Tsc2 fl/fl Lyz2 -Cre mouse at day 0 and 14 of everolimus treatment. ( d ) IHC for Mac-2 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus. ( e ) IHC for p-S6 and cleaved caspase 3 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus for two days. Representative histological images are from one mouse; each analysis was performed on four ( a , b , c ) or three ( d , e ) mice per genotype. Scale bar, 40 μm ( a ), 100 μm ( b ), 1 mm ( d ), 50 μm ( e ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining

Journal: Scientific reports

Article Title: Nox4 is a Target for Tuberin Deficiency Syndrome.

doi: 10.1038/s41598-018-21838-4

Figure Lengend Snippet: Figure 1. ROS levels are increased in human angiomyolipomas and in Tsc2+/− mouse model. (A) H2O2 levels and (B) Superoxide production in renal angiomyolipoma samples (AML) from TSC patients (n = 8) and normal kidney biopsy (n = 17) were shown, measured by Amplex red assay and lucigenin chemiluminescence, respectively. (C) H2O2 levels and (D) Superoxide production in control (n = 6) and Tsc2+/− mouse (n = 10) kidneys was measured by Amplex red and lucigenin chemiluminescence assays, respectively. (E) ROS levels were measured by flow cytometry with DCF dye in proximal tubular epithelial cells with siScramble (orange and light blue) or siTSC2 (green and dark blue) (left panel). The top right panel shows the western blotting for tuberin protein levels in these cells. The bottom right panel showed the quantification of the data for DCF dye measurement. (F) ROS levels were measured with DHE dyes, scale bar: 200 μm and the intensity of staining per cells were quantified (right panel). Mean ± SE of 3 independent experiment, *p < 0.05 vs siScr; (G) Superoxide production was measured in siScramble or siTSC2 transfected cells by lucigenin chemiluminescence. The data are presented as the mean ± S.E of 3 repeats. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with normal human kidney (A,B), or control mice (C,D), or cells receiving scramble siRNA (E–G).

Article Snippet: Lentiviral pLKO.1-puro vectors encoding shRNA specific for TSC2 (#15478) or control scramble (#1864) were purchased 1 2SCIENTIfIC RepoRts | (2018) 8:3781 | DOI:10.1038/s41598-018-21838-4 from Addgene (Cambridge, MA).

Techniques: Amplex Red Assay, Control, Flow Cytometry, Western Blot, Staining, Transfection, Comparison

Journal: Scientific reports

Article Title: Nox4 is a Target for Tuberin Deficiency Syndrome.

doi: 10.1038/s41598-018-21838-4

Figure Lengend Snippet: Figure 2. Tuberin regulates Nox4 protein expression. (A) RT-PCR analysis of the Nox isoforms in human proximal tubular epithelial cells with primers listed in Supplementary Table 1. (B) Nox4 protein levels were determined by western blotting in human proximal tubular epithelial cells transfected with Scramble or TSC2 siRNA; (C) mRNA levels were measured by qRT-PCR in cells transfected with Scramble or TSC2 siRNA; The data are presented as the mean ± S.E, in comparison with cells receiving scramble siRNA (n = 4). (D) Proximal tubular epithelial cells stably expressing TSC2 shRNA were infected with adenoviruses expressing GFP or TSC2; tuberin and Nox4 protein levels were measured in these cells by western blotting. (E) Nox4 RNA stability was measured by qRT-PCR in Actinomycin D (5 μg/mL)-treated shTSC2 cells. (F) Protein stability of Nox4 was measured by western blotting using cycloheximide (CHX) (100 μg/mL)-treated shTSC2 cells for indicated times. Lower panel showed the percentage changes of protein during the CHX treatment; (G) Nox4 and tuberin protein levels were measured in RK3E and LEF2 cells by western blotting; (H) Nox4 and tuberin protein levels were measured in LEF2 cells infected with adenoviruses containing GFP or TSC2; (I) Nox4 and tuberin protein levels were measured in control and Tsc2+/− mouse kidney cortices; (J) Nox4 protein levels were measured in renal angiomyolipoma samples from TSC patients and normal human kidney biopsies. In all western blotting assays, β-actin or GAPDH serves as loading controls.

Article Snippet: Lentiviral pLKO.1-puro vectors encoding shRNA specific for TSC2 (#15478) or control scramble (#1864) were purchased 1 2SCIENTIfIC RepoRts | (2018) 8:3781 | DOI:10.1038/s41598-018-21838-4 from Addgene (Cambridge, MA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Quantitative RT-PCR, Comparison, Stable Transfection, shRNA, Infection, Control

Journal: Oncotarget

Article Title: RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer

doi:

Figure Lengend Snippet: (A) and (B) Western blotting of PKR, phosphorylated (p-)PKR, p-eIF2α, AMPK, p-AMPK T172 , p-AMPK T485 , tuberous sclerosis complex 2 (TSC2), p-TSC2, acetyl-CoA carboxylase (ACC), p-ACC, mammalian target of rapamycin (mTOR), and p-mTOR protein expression in human lung cancer cell lines (H1299, A549, and H322) 72 hours after transfection with PBS (control [con]), Ad-PKR (2,500 viral particles/cell), Ad-PKRΔ6 (2,500 viral particles/cell), or Ad-Luc (2,500 viral particles/cell). The expression of actin was used as a loading control.

Article Snippet: We obtained the following antibodies from Cell Signaling Technology: AMPK (2532), phospho-AMPK α (Thr172) (40H9) (2535), p-TSC2 (Ser1387) (5584), acetyl-CoA carboxylase (C83B10) (3676), phospho-acetyl-CoA carboxylase (Ser79) (3661), phospho-mTOR (Ser2448) (5536), phospho-LKB1 (Ser428) (C67A3) (3482), TAK1 (4505), and phospho-TAK1 (Thr184/187) (90C7) (4508).

Techniques: Western Blot, Expressing, Transfection, Control