Journal: JHEP Reports

Article Title: Downregulation of hepatic METTL3 contributes to APAP-induced liver injury in mice

doi: 10.1016/j.jhepr.2023.100766

Figure Lengend Snippet: Hepatic deficiency of Mettl3 decreases proteasome activity, which contributes to the increased protein stability of MKK4 and JNK. (A and B) RNA-seq analysis was performed in the livers of Mettl3 flox/flox and Mettl3- HKO mice at 8 weeks old (n = 3 for each group). KEGG pathway analysis of the downregulated genes in Mettl3- HKO mice (A). A heatmap of downregulated proteasome-related genes is shown (B). (C and D) Proteasome activity (n = 8 mice for each group, p = 0084; Student's t tests) and PSMB4/6 protein levels (n = 4 mice for each group; PSMB4, p = 0.0002; PSMB6, p = 0.0009; Student's t tests) were measured in the livers of Mettl3 flox/flox and Mettl3- HKO mice. (E and F) Primary hepatocytes were isolated from Mettl3 -HKO and Mettl3 flox/flox mice and then infected with Ad-βGal, Ad-FLAG-PSMB4, and Ad-FLAG-PSMB6 adenovirus. MKK4, JNK, FLAG, METTL3, and Tubulin protein levels were measured by immunoblotting and quantified by ImageJ (n = 3 independent samples for each group; for MKK4 in (E), Mettl3 -HKO + βGal vs . Mettl3 flox/flox +βGal, p = 0.0381; Mettl3 -HKO + βGal vs . Mettl3 -HKO + FLAG-PSMB4, p = 0.0466; for JNK in (E), Mettl3 -HKO + βGal vs . Mettl3 flox/flox + βGal, p = 0.0121; Mettl3 -HKO + βGal vs . Mettl3 -HKO+ FLAG-PSMB4, p = 0.0479; for MKK4 in (F), Mettl3 -HKO + βGal vs . Mettl3 flox/flox + βGal, p <0.0001; Mettl3 -HKO + βGal vs . Mettl3 -HKO+ FLAG-PSMB6, p = 0.0051; for JNK in (F), Mettl3 -HKO + βGal vs . Mettl3 flox/flox + βGal, p <0.0001; Mettl3 -HKO + βGal vs . Mettl3 -HKO + FLAG-PSMB6, p = 0.0014; Student's t tests). These experiments were repeated three times independently with similar results. Differences between two groups were analysed using Student's t tests. ∗ p <0.05. ∗∗ p <0.01. Data represent the mean ± SEM. KEGG, Kyoto Encyclopedia of Genes and Genomes; Mettl3 -HKO, hepatocyte-specific Mettl3 knockout; METTL3, methyltransferase-like 3; RNA-seq, RNA sequencing.

Article Snippet: Antibodies were as follows: FLAG (F1804, Sigma), JNK1 (A0288, Abclonal), JNK2 (A1251, Abclonal), JNK (9252, Cell Signaling Technology), p-JNK (9251, Cell Signaling Technology), p-MKK4 (9156, Cell Signaling Technology), MKK4 (9152, Cell Signaling Technology), METTL3 (96391, Cell Signaling Technology), PSMB4 (11029-1-AP, Proteintech), PSMB6 (11684-2-AP, Proteintech), CYP2E1 (19937-1-AP, Proteintech), Tubulin (sc5286, Santa Cruz), AIF (17984-1-AP, Proteintech), EndoG (22148-1-AP, Proteintech), and cytochrome c (Cytc) (ab13575, Abcam).

Techniques: Activity Assay, RNA Sequencing, Isolation, Infection, Western Blot, Knock-Out

Journal: JHEP Reports

Article Title: Downregulation of hepatic METTL3 contributes to APAP-induced liver injury in mice

doi: 10.1016/j.jhepr.2023.100766

Figure Lengend Snippet: METTL3 decreases MKK4 and JNK protein levels by upregulating PSMB4 and PSMB6, which protects hepatocytes from APAP-induced hepatocyte death. (A) Primary hepatocytes were isolated from C57BL/6 WT mice and then infected with Ad-βGal and Ad-FLAG-METTL3 adenovirus overnight. MKK4, JNK, PSMB4, PSMB6, FLAG, and Tubulin protein levels were measured by immunoblotting (n = 3 for each group). (B) Hepa1-6 cells were infected with Ad-βGal and Ad-FLAG-METTL3 adenovirus overnight. MKK4, JNK, PSMB4, PSMB6, FLAG, and Tubulin protein levels were measured by immunoblotting (n = 3 for each group). (C) Hepa1-6 cells were infected with Ad-βGal and Ad-FLAG-METTL3 adenovirus overnight and then treated with or without APAP for 4 h. PI staining was used to measure cell death (n = 5 for each group; Control, p = 0.8507; APAP, p = 0.0001; Student's t tests). (D and E) Hepa1-6 cells were infected with Ad-Scramble, Ad-sh Mettl3 -1, and Ad-sh Mettl3 -2 adenovirus for 24 h and then infected with Ad-βGal and Ad-FLAG-METTL3 adenovirus overnight. Some of the cells were used for measuring MKK4, JNK, PSMB4, PSMB6, FLAG, and Tubulin protein levels by immunoblotting (D) (n = 3 for each group). Some of the cells were treated with or without APAP for 4 h. PI staining was used to measure cell death (E) (n = 5 for each group; Control: βGal + sh Mettl3 -1 vs . Scramble, p = 0.8283; βGal + sh Mettl3 -2 vs . Scramble, p = 0.5396; sh Mettl3 -1 + FLAG-METTL3 vs . Scramble, p = 0.3924; sh Mettl3 -2 + FLAG-METTL3 vs . Scramble, p = 0.8175; APAP: βGal + sh Mettl3 -1 vs . Scramble, p = 0.0003; βGal + sh Mettl3 -2 vs . Scramble, p = 0.0043; βGal + sh Mettl3 -1 vs . sh Mettl3 -1 + FLAG-METTL3, p = 0.0009; βGal + sh Mettl3 -2 vs . sh Mettl3 -2 + FLAG-METTL3, p <0.0001; ANOVA, LSD t test). ∗ p <0.05. ∗∗ p <0.01. Data represent the mean ± SEM. APAP, acetaminophen; DIC, differential interference contrast; Mettl3 -HKO, hepatocyte-specific Mettl3 knockout; METTL3, methyltransferase-like 3; PI, propidium iodide; WT, wild-type.

Article Snippet: Antibodies were as follows: FLAG (F1804, Sigma), JNK1 (A0288, Abclonal), JNK2 (A1251, Abclonal), JNK (9252, Cell Signaling Technology), p-JNK (9251, Cell Signaling Technology), p-MKK4 (9156, Cell Signaling Technology), MKK4 (9152, Cell Signaling Technology), METTL3 (96391, Cell Signaling Technology), PSMB4 (11029-1-AP, Proteintech), PSMB6 (11684-2-AP, Proteintech), CYP2E1 (19937-1-AP, Proteintech), Tubulin (sc5286, Santa Cruz), AIF (17984-1-AP, Proteintech), EndoG (22148-1-AP, Proteintech), and cytochrome c (Cytc) (ab13575, Abcam).

Techniques: Isolation, Infection, Western Blot, Staining, Control, Knock-Out

Journal: JHEP Reports

Article Title: Downregulation of hepatic METTL3 contributes to APAP-induced liver injury in mice

doi: 10.1016/j.jhepr.2023.100766

Figure Lengend Snippet: METTL3 interacts with RNA polymerase II (Rpb1 CTD) and active histone modifications such as H3K9ac, H3K27ac, and H3K36me3, which is required for maintaining the expression of proteasome-related genes. (A) Primary hepatocytes were isolated from Mettl3 -HKO and Mettl3 flox/flox mice and then treated with actinomycin D (ACTD) (10 μg/mL) for different time points (0, 3, 6, and 12 h). Psmb4 and Psmb6 mRNA levels were measured by RT-qPCR (n = 6 independent cell samples for each group; Psmb4 : 3 h, p = 0.3232; 6 h, p = 0.6135; 12 h, p = 0.1166; Psmb6 : 3 h, p = 0.6464; 6 h, p = 0.1429; 12 h, p = 0.012; Student's t tests). (B) The fractions of 40s, 60s, 80s, and polysomes in the livers of Mettl3 -HKO and Mettl3 flox/flox mice were identified by sucrose gradient centrifugation. The relative distribution of mRNAs of Psmb4 and Psmb6 in these fractions was measured by semiquantitative RT-PCR (n = 3 mice for each group; Psmb4 : 40s, p = 0.9242; 60s, p = 0.0454; 80s, p = 0.6224; polysome: p = 0.7855; p = 0.4753; p = 0.4564; p = 0.1707; Psmb6 : 40s, p = 0.5247; 60s, p = 0.7162; 80s, p = 0.5408; polysome: p = 0.8025; p = 0.2729; p = 0.7528; p = 0.9169; Student's t tests). (C) Combined analysis of the ATAC-seq and RNA-seq data showed that five proteasome-related genes, namely, Psma6 , Psmb3 , Psmb4 , Psmb6 and Gm13835 , were downregulated in Mettl3- HKO mice. (D) Primary hepatocytes were isolated from C57BL/6 WT mice and then infected with βGal and FLAG-METTL3 adenovirus. Total cell lysates were incubated with DNase1 (200 U/ml) at 37 °C for 30 min. These cell lysates were immunoprecipitated with FLAG beads and then immunoblotted with anti-H3K9ac, anti-H3K27ac, anti-H3K36me3, anti-Rpb1 CTD, and anti-FLAG antibodies. This experiment was repeated twice independently with similar results. (E) RNA polymerase II (Rpb1 CTD) levels in the promoters of Psma6 , Psmb3 , Psmb4 , Psmb6 , and Actb genes in the livers of Mettl3 flox/flox and Mettl3- HKO mice at 8 weeks old (n = 5 mice for each group; Psma6 , p = 0.0256; Psmb3 , p = 0.027; Psmb4 , p = 0.0024; Psmb6 , p = 0.0033; Actb , p = 0.3224; Student's t tests). Differences between two groups were analysed using Student's t tests. ∗ p <0.05. ∗∗ p <0.01. Data represent the mean ± SEM. ACTD, actinomycin D; ATAC-seq, assay for transposase-accessible chromatin with sequencing; Mettl3 -HKO, hepatocyte-specific Mettl3 knockout; METTL3, methyltransferase-like 3; RNA-seq, RNA sequencing; RT-PCR, real-time PCR; RT-qPCR, real-time quantitative PCR; WT, wild-type.

Article Snippet: Antibodies were as follows: FLAG (F1804, Sigma), JNK1 (A0288, Abclonal), JNK2 (A1251, Abclonal), JNK (9252, Cell Signaling Technology), p-JNK (9251, Cell Signaling Technology), p-MKK4 (9156, Cell Signaling Technology), MKK4 (9152, Cell Signaling Technology), METTL3 (96391, Cell Signaling Technology), PSMB4 (11029-1-AP, Proteintech), PSMB6 (11684-2-AP, Proteintech), CYP2E1 (19937-1-AP, Proteintech), Tubulin (sc5286, Santa Cruz), AIF (17984-1-AP, Proteintech), EndoG (22148-1-AP, Proteintech), and cytochrome c (Cytc) (ab13575, Abcam).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Infection, Incubation, Immunoprecipitation, Sequencing, Knock-Out, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A) Western blots for ribosomal protein subunits RPL5, RPL7, RPL11, and RPL26 in WiT49 and 17.94 following 48-hour treatment of DMSO, 2nM actD, 8nM BTZ, or 2nM ActD + 8nM BTZ. (B) Protein levels of proteasome subunits PSMA6 (α1), PSMB1 (β6), PSMB2 (β4), PSMB5 (β5), and PSMD1 (P112), and POMP in WiT49 and 17.94 following 18-, 24-, 48-, and 72-hour 2nM actD treatment versus vehicle (DMSO). (C) Quantification of relative proteasome-specific chymotrypsin-like activity in WiT49 and 17.94 cells following 24 hours of DMSO versus 2nM actD, 8nM BTZ, or 2nM actD + 8nM BTZ treatments (Student’s t-test p-value versus vehicle: *<0.05, ** <0.01, ****<0.0001).

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A) Top KEGG pathways enriched in RNA-seq of DAWT versus FHWT. (B) Top reactome pathways enriched in RNA-seq of DAWT versus FHWT. (C,D) Overall survival of tumors stratified according to expression of proteasome enzymatic subunit genes PSMB1, PSMB2, PSMB5 among patients with favorable histology tumors (C) or anaplastic histology (D) . (Log Rank test p-value: *<0.05, ** 0.01)

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A,B) GSEA plot showing enrichment for KEGG_PROTEASOME (A) and REACTOME_APC_C_MEDIATED_DEGRADATION_OF_CELL_CYCLE_PROTEINS (B) in anaplastic Wilms tumor vs. favorable-histology Wilms tumors in TARGET dataset. (C) Overall survival of Wilms tumors, regardless of histology, stratified according to expression of proteasome enzymatic subunit genes PSMB1, PSMB2, PSMB5 among patients (Log rank test p-value: *<0.05, *** 0.001). (D) Model for effect of actinomycin D on protein homeostasis in Wilms tumor cells.

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: Wilms Tumor Assay, Expressing

Journal: JCI Insight

Article Title: ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5

doi: 10.1172/jci.insight.157081

Figure Lengend Snippet: ( A ) Molecular structure of biotinylated BTZ (BTZ-b). ( B ) Flow cytometry–based apoptosis assay to examine the cytotoxicity of BTZ-b in CTR-KD versus ISG-KD (sh1) ARD cells. The cells were treated with BTZ-b (2 μM) for 24 hours. DMSO- or biotin-treated cells served as CTRs ( n = 3). Student’s t test was performed. * P ≤ 0.05. ( C ) BTZ-b pull-down assay using ARD cell lysate in the presence of BTZ to examine BTZ competition with BTZ-b in PSMB5 binding. BTZ was added to the cell lysate to a final concentration of 200 μM and incubated with BTZ-b–coated beads for competition. ( D ) Biotinylated BTZ pull-down assay using CTR-KD and ISG-KD ARD cell lysates. Biotin served as a negative control for pull-down. The pull-down precipitate and the whole-cell lysates (input) were subjected to Western blotting. ( E ) BTZ-b pull-down assay using CTR and ISG-OE AMO1 cell lysates. ( F ) BTZ-b pull-down assay using recombinant ISG20L2 (rISG) and PSMB5 (rB5) proteins. The recombinant protein was dissolved in PBS. The final concentration of rB5 was 0.5 μg/mL (+), while the final concentration of rISG was 0.25 μg/mL (+) or 1.5 μg/mL (++). Using the gray intensity of PSMB5 pull-down by BTZ-b alone as a control, ImageJ software (NIH) was used to determine the levels of PSMB5 pull-down by BTZ-b with rISG competition. Numbers indicated on the lane. ( G ) BTZ binds to ISG20L2 as shown by SPR measurements. Graphs of equilibrium response unit (RU) responses versus compound concentrations were plotted. The estimated K D is 26.71 μM, as shown by the vertical dotted line. ( H ) Schematic mechanism of ISG20L2-induced MM BTZ resistance.

Article Snippet: Recombinant human ISG20L2 protein was custom-made by Merry Bio Technology Co., Ltd. Western blot antibodies against PSMB5 (catalog sc-393931), PSMD8 (catalog sc-514053), PSMD3 (catalog sc-393588), PSMC5 (catalog sc-390631), and eIF2a (catalog sc-133132) were purchased from Santa Cruz Biotechnology.

Techniques: Flow Cytometry, Apoptosis Assay, Pull Down Assay, Binding Assay, Concentration Assay, Incubation, Negative Control, Western Blot, Recombinant, Control, Software

Journal: bioRxiv

Article Title: Molecular aging is the main driver of Parkinson’s Disease

doi: 10.1101/2025.08.27.672359

Figure Lengend Snippet: (A-C) Determination of proteasomal activity of Caspase-like enzymes of full-brain lysates from S1/S2 animals by proteasome activity assay. Comparison of short-, long- and non-expressing animals with (A) 6 M, (B) 16 M and (C) 24 M of age. Luminescence signal (counts per second, CPS) is proportional to proteasomal activity and was normalized to total amount of 20S proteasome determined by Western blot analysis (student’s t-test, ***<0.001, **<0.01; data: mean ± SEM). (D) Top 20 PD Signature genes and 20 randomly sampled genes were used to calculate a Module Score on the human midbrain snRNA-seq dataset Smajic et al., 2022. P values were calculated by Wilcoxon test on sample wise pseudo-bulked samples. Reflecting our finding in mice GABA-Rgs9 Neurons (GABAergic BGNs), the PD Signature is found to be most affecting the GABAergic Neurons in the human midbrain. This effect is not observed in the random control. (E) Heatmap of scaled log2 Fold Changes of genes from the PD Signature that were concordantly changed across mice GABA-Rgs9 synON24M vs synOFF24M, Smajic et al., human PD vs Control in GABAergic neurons, and Martirosyan et al. PD vs Control in GABAergic neurons. We obtained a total of 53 concordant genes, including genes we deemed as central to our PD Signature, such as Ubb, Dynll1, or Calm1.

Article Snippet: For Western blots with the antibody against the 20S proteasome α1/α2/α3/α5/α6/α7 subunits (Santa Cruz), only 12 % polyacrylamide gels and 40 μg protein per sample was used.

Techniques: Activity Assay, Comparison, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: 6-bromo-indirubin-3′-oxime (6BIO), a Glycogen synthase kinase-3β inhibitor, activates cytoprotective cellular modules and suppresses cellular senescence-mediated biomolecular damage in human fibroblasts

doi: 10.1038/s41598-017-11662-7

Figure Lengend Snippet: Treatment of human skin fibroblasts with 6BIO activated proteostatic modules. ( A 1 – A 3 ) Relative expression of molecular chaperones genes ( hsp27 , hspa1b/hsp70-2 , clu , stub1 ) ( A 1 ), of genes involved in ALP functionality ( becn1 , sqstm1 , hdac6 , ctsl , ctsd ) ( A 2 ), as well as of proteasomal genes ( psma7 , psmb1 , psmb2 , psmb5 , rpn6 , rpn11 ) ( A 3 ), in cells treated with the indicated concentrations of 6BIO for 24 h. ( B ) Representative immunoblot analyses of the autophagy-related Beclin1 and the 26S proteasome subunits Rpn6, Rpt6, α7 and β5 expression levels after cell exposure to the shown concentrations of 6BIO for 36 h. ( C ) Relative proteasome peptidase activities (LLVY/β5, LLE/β1 and LRR/β2) in cells treated with 1 or 2 μM 6BIO for 36 h. The beta-2-microglobulin ( b2m ) gene expression ( A 1 – A 3 ) and GAPDH probing ( B ) were used as reference for RNA and protein input, respectively. In ( A 1 – A 3 ) control samples values were set to 1; in ( C ) control values were set to 100%. Quantitation of shown blots is presented in Suppl. Fig. . Bars, ± SD; *P < 0.05; **P < 0.01.

Article Snippet: Primary antibodies against Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Beclin1 (sc-11427), Gsk-3β (sc-9166), p53 (sc-47698), p21 WAF1/CIP1 (sc-6246), Actin (sc-1615) and GAPDH (sc-25778), as well as the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Gene Expression, Control, Quantitation Assay

Journal: Scientific Reports

Article Title: 6-bromo-indirubin-3′-oxime (6BIO), a Glycogen synthase kinase-3β inhibitor, activates cytoprotective cellular modules and suppresses cellular senescence-mediated biomolecular damage in human fibroblasts

doi: 10.1038/s41598-017-11662-7

Figure Lengend Snippet: siRNA-mediated gsk-3β gene expression knock down in human fibroblasts largely phenocopied the 6BIO-induced effects. ( A 1 ) Q-RT-PCR assay of gsk-3β gene expression levels after transfecting (or not) cells with Gsk-3β RNAi oligonucleotides for 24 h. ( A 2 ) Representative immunoblot analyses of protein samples probed with antibodies against Gsk-3β and p-Gsk-3 Ser21/9 after Gsk-3β RNAi for 48 h. ( B 1 ) Q-PCR expression analyses of Nrf2 transcriptional target genes ( keap1 , nqo1 , txnrd1 ) after Gsk-3β RNAi for 24 h. ( B 2 ) Immunoblotting analysis of Nrf2 expression after Gsk-3β RNAi for 24 h. ( C 1 ) Relative expression of 20S ( psmb1 , psmb5 ) or 19S ( rpn11 ) proteasome genes after Gsk-3β RNAi for 24 h. ( C 2 ) Representative immunoblot analyses of protein samples probed with antibodies against the 20S proteasome subunits α7 and β5 after Gsk-3β RNAi for 48 h. Probing with GAPDH was used in ( A 2 , B 2 , C 2 ) as total protein loading reference; b2m gene expression ( A 1 , B 1 , C 1 ) was used as reference for RNA input. Quantitation of shown blots is presented in Suppl. Fig. . Bars, ± SD; * P < 0.05; ** P < 0.01 vs . controls set to 1.

Article Snippet: Primary antibodies against Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Beclin1 (sc-11427), Gsk-3β (sc-9166), p53 (sc-47698), p21 WAF1/CIP1 (sc-6246), Actin (sc-1615) and GAPDH (sc-25778), as well as the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Gene Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Quantitation Assay

Journal: Scientific Reports

Article Title: 6-bromo-indirubin-3′-oxime (6BIO), a Glycogen synthase kinase-3β inhibitor, activates cytoprotective cellular modules and suppresses cellular senescence-mediated biomolecular damage in human fibroblasts

doi: 10.1038/s41598-017-11662-7

Figure Lengend Snippet: Long-term treatment of normal human fibroblasts with 6BIO significantly reduced the senescence-mediated increase in oxidative and proteotoxic stress. ( A ) Relative expression of proteasome genes ( psma7 , psmb1 , psmb2 , psmb5 , rpn6 , rpn11 ) after long-term cell treatment with 6BIO; in the upper insert the expression levels of the proteasomal subunits α7 and β5 after exposing cells to 6BIO for 25 days are shown. ( B ) Confocal analysis of cells incubated with 6BIO for 25 days after labelling with an antibody against Nrf2 and counterstaining with DAPI. ( C ) CLSM ROS detection in BJ cells after treatment with 6BIO for 25 days. ( D ) Lipofuscin fluorescence imaging after long-term treatment of BJ cells with 6BIO. (E) Representative CLSM images following detection of the Ser 139 phosphorylated form of histone H 2 A.X (γH 2 A.X) after long-term treatment of cells with 6BIO. Cells nuclei were counterstained with DAPI (blue) and actin with Phalloidin (red). The b2m gene expression ( A ) was used as a total RNA loading reference. In all cases cells were treated with 1 μM 6BIO. Quantitation of shown blots is presented in Suppl. Fig. . Bars, ± SD; *P < 0.05; **P < 0.01 vs . controls set to 1. Bars, in ( B , C , E ) 10 μM, and in ( D ) 50 μM.

Article Snippet: Primary antibodies against Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Beclin1 (sc-11427), Gsk-3β (sc-9166), p53 (sc-47698), p21 WAF1/CIP1 (sc-6246), Actin (sc-1615) and GAPDH (sc-25778), as well as the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Incubation, Fluorescence, Imaging, Gene Expression, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation

doi: 10.1074/jbc.RA119.010796

Figure Lengend Snippet: Roseltide rT7 is a human 20S proteasome inhibitor. A, representative Western blotting analysis on the ubiquitinated protein expressions in A549 cells for 2 h with 10 μm roseltide rT7; 1 μm MG132 was used as positive control. B, dose-response effect of roseltide rT7 and MG132 on human 20S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 20S chymotrypsin-like proteasome activities are 3.3 ± 0.04 and 0.13 ± 0.03 μm, respectively. C, in-gel fluorescence image of human 20S proteasome treated with roseltide rT7 or MG132 for 1 h followed by 30 min with Me4BodipyFL-Ahx3Leu3VS probe. D, MALDI-TOF MS profiles of roseltide rT7 (10 μm) without (upper panel) or with human 20S proteasome (5 nm) (lower panel) for 24 h at 37 °C. E, dose-response effect of roseltide rT7 and MG132 on human 26S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 26S chymotrypsin-like proteasome activities are 14.93 ± 0.04 and 0.05 ± 0.02 μm, respectively. F, effects of linear pentapeptides, IIMLP, IIMLA, IIMLK, IIMPA, and IIMPK, on human 20S chymotrypsin-like proteasome activities using a Proteasome-GloTM chymotrypsin-using assay kit. The proteasome cleavage of P1′-P1 site was determined using RP-HPLC and LC-MS analysis. P(X) refers to N-terminal to the substrate cleavage site. P(X)′ refers to C-terminal to the substrate cleavage site.

Article Snippet: Briefly, purified human 20S proteasome (100 p m ; BostonBiochem) or human 26S proteasome (500 p m ; BostonBiochem) was incubated with MG132 (Santa Cruz Biotechnology), roseltide rT7, or pentapeptide analogs in 10 m m HEPES buffer (pH 7.6).

Techniques: Western Blot, Positive Control, Fluorescence, Liquid Chromatography with Mass Spectroscopy

Journal: Virology Journal

Article Title: HSV-2 glycoprotein gD targets the CC domain of tetherin and promotes tetherin degradation via lysosomal pathway

doi: 10.1186/s12985-016-0610-7

Figure Lengend Snippet: HSV-2 gD downregulates tetherin via lysosomal pathway. a HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag/gM-flag were costained with anti-flag (red), anti-tetherin (purple) and anti-cathepsin D (green) antibodies. Nuclei were counterstained with Hoechst 33258 (blue). The colocalization of tetherin with lysosome markers (cathepsin D) was assessed by confocal microscopy. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm. b HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag/gM-flag were costained with anti-flag (red), anti-tetherin (purple) and anti-20S proteasome (green) antibodies. Nuclei were counterstained with Hoechst 33258 (blue). The colocalization of tetherin with the proteasome markers (20S proteasome) was assessed by confocal microscopy. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm. c Pearson’s correlation coefficients were analyzed to determine the colocalization of tetherin and cathepsin D. d Pearson’s correlation coefficients were analyzed to determine the colocalization of tetherin and 20S proteasome. Data shown are mean ± SD by quantitative analyses of at least 20 distinct cells. e Western blot was used to analyze the expression of total tetherin in HeLa cells transfected with control plasmid and plasmid expressing gD-flag (treated or untreated with lysosome protease inhibitors (LPI)) where actin was used as a loading control. f HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag were treated by lysis buffer. The prepared cell extract was ultracentrifuged by density gradient centrifugation and the lysosome band is located in the top 2 mL of the gradient. The corresponding bands were collected and the finally harvested lysosome pellets were detected by western blot. Samples 1 and 2 were two finally harvested lysosome pellets in two representative experiments

Article Snippet: Thereafter, cells were incubated for 1 h at 37 °C with mouse monoclonal antibody against tetherin (H00000684-M15, Abnova) at a dilution of 1:200 and rabbit polyclonal antibody against cathepsin D (sc-10725; Santa Cruz) (or 20S proteasome α7/α8 (sc-67344; Santa Cruz)) at a dilution of 1:100 in PBS-2 % (w/v) BSA, followed by incubation for 1 h at 37 °C with Cy5-conjugated goat anti-mouse secondary antibody (Beyotime, China), FITC-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at a dilution of 1:200 in PBS-2 % (w/v) BSA.

Techniques: Transfection, Plasmid Preparation, Expressing, Confocal Microscopy, Western Blot, Control, Lysis, Gradient Centrifugation