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TRIM44 enhances autophagy via SQSTM1 oligomerization in response to oxidative stress

Wang, Yuqin, Lyu, Lin, Vu, Trung, McCarty, Nami·2024·Scientific Reports
DOI 10.1038/s41598-024-67832-xPMC PMC11329658
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Figure 1 TRIM44 attenuates cytotoxicity via enhancing autophagy in response to oxidative stress. (A) Cell survival assay. U266 TRIM44[OE-CON] and TRIM44[OE] cells were challenged with different concentrations of As[III] as indicated for 24 h. Percentage of cell survival was determined by cell counting with trypan blue staining. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01 (unpaired t-tests). (B) Cell survival assay. U266 TRIM44[KD-CON] and TRIM44[KD] cells were challenged with different concentrations of As[III] as indicated for 24 h. Percentage of cell survival was determined by cell counting with trypan blue staining. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01 (unpaired t-tests). (C) Flow cytometry analysis. U266 cells were treated with or without As[III] (5 μM, 24 h) and then subjected to be stained with PE/7-AAD. Percentage of apoptosis was quantified. **P < 0.01, ***P < 0.001 (unpaired t-tests). (D) Immunoblot analysis. U266 cells were treated with or without As[III] (5 μM) for 24 h. Cell lysates were then analyzed by immunoblotting for LC3B-II. GAPDH was used as a loading control. (E) Flow cytometry analysis. U266 cells were co-treated with or without As[III] (5 μM) and autophagy inhibitor 3-Methyladenine (3-MA, 500 μM) for 24 h. and then subjected to be stained with PE/7-AAD. Percentage of apoptosis was quantified. **P < 0.01, ***P < 0.001 (unpaired t-tests).
Figure 1 TRIM44 attenuates cytotoxicity via enhancing autophagy in response to oxidative stress. (A) Cell survival assay. U266 TRIM44[OE-CON] and TRIM44[OE] cells were challenged with different concentrations of As[III] as indicated for 24 h. Percentage of cell survival was determined by cell counting with trypan blue staining. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01 (unpaired t-tests). (B) Cell survival assay. U266 TRIM44[KD-CON] and TRIM44[KD] cells were challenged with different concentrations of As[III] as indicated for 24 h. Percentage of cell survival was determined by cell counting with trypan blue staining. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01 (unpaired t-tests). (C) Flow cytometry analysis. U266 cells were treated with or without As[III] (5 μM, 24 h) and then subjected to be stained with PE/7-AAD. Percentage of apoptosis was quantified. **P < 0.01, ***P < 0.001 (unpaired t-tests). (D) Immunoblot analysis. U266 cells were treated with or without As[III] (5 μM) for 24 h. Cell lysates were then analyzed by immunoblotting for LC3B-II. GAPDH was used as a loading control. (E) Flow cytometry analysis. U266 cells were co-treated with or without As[III] (5 μM) and autophagy inhibitor 3-Methyladenine (3-MA, 500 μM) for 24 h. and then subjected to be stained with PE/7-AAD. Percentage of apoptosis was quantified. **P < 0.01, ***P < 0.001 (unpaired t-tests).CC BY-NC-ND · PubMed Central
Figure 2 TRIM44 promotes SQSTM1 oligomerization. (A) The domain structure of TRIM44 protein. Depicts full-length (FL) TRIM44 (aa 1-344), ΔZF truncate (aa 48–344). (B) Confocal microscopy of HeLa cells co-transfected with HA-SQSTM1 and GFP, or GFP-TRIM44 full-length or GFP-TRIM44ΔZF. Immunostaining with HA antibody was done after 48 h. Scale bars represent 10 μm. (C) Graph illustrating the average volume of HA-SQSTM1 oligomerization. Average volume from 50 cells counted for each data point (bar). Statistical analysis via one-way ANOVA with Tukey's test, **P < 0.01, ***P < 0.001. (D) Confocal microscopy of HeLa cells co-transfected with GFP or GFP-TRIM44 and specific HA-SQSTM1 constructs (HA-SQSTM1, HA-SQSTM1K7R, HA-SQSTMQD69A, and HA-SQSTM1K7AD69A). Immunostaining with HA antibody performed post 48 h. Scale bars: 10 µm. (E) Graph of average volume of HA-SQSTM1 constructs oligomerization (HA-SQSTM1, HA-SQSTM1K7R, HA-SQSTM1D69A, and HA-SQSTM1K7AD69A). One-way ANOVA with Tukey's test for statistical analysis. ***P < 0.001. Black star (*) denotes GFP-TRIM44 transfected cells versus corresponding GFP transfected cells. Red star (*) denotes HA-SQSTM1 mutants’ transfected cells versus corresponding wild-type HA-SQSTM1 transfected cells. (F) In vivo deubiquitylation assay. 293T cells were co-transfected with HA-SQSTM1 constructs (HA-SQSTM1 and HA-SQSTM1K7AD69A), His-Ubiquitin and GFP-TRIM44 plasmids. Post 48 h, cells were treated with proteasome inhibitor MG132 (5 μM, 6 h), followed by immunoprecipitation with HA antibody. Anti-Ub antibody was used to immunoblot ubiquitinated HA-SQSTM1 and HA-SQSTM1K7AD69A. (G) HeLa cells were transfected with FLAG-HA-SQSTM1 or FLAG-HA-SQSTM1ΔPB1 mutant (lacking PB1 domain), in conjunction with either GFP or GFP-TRIM44. After 48 h, immunostaining was performed with HA (red) and DRAQ5 (blue). Scale bars represent 10 μm. (H) Graphic presentation of the average oligomerization volume of SQSTM1 (FLAG-HASQSTM1, FLAG-HA-SQSTM1ΔPB1). Each bar reflects the oligomerization volume of the indicated HA-SQSTM1 constructs in 50 cells. Statistical analysis was carried out using one-way ANOVA with Tukey's test, with **P < 0.01 and ***P < 0.001. (I) 293T cells were co-transfected with the indicated plasmids. After 48 h, cells were cross-linked with DSP (0.4 mg/ml, 4 °C, 2 h) and processed under either reducing (with β-ME) or nonreducing conditions (without β-ME). The lysates were then subjected to immunoblotting using the indicated antibodies.
Figure 2 TRIM44 promotes SQSTM1 oligomerization. (A) The domain structure of TRIM44 protein. Depicts full-length (FL) TRIM44 (aa 1-344), ΔZF truncate (aa 48–344). (B) Confocal microscopy of HeLa cells co-transfected with HA-SQSTM1 and GFP, or GFP-TRIM44 full-length or GFP-TRIM44ΔZF. Immunostaining with HA antibody was done after 48 h. Scale bars represent 10 μm. (C) Graph illustrating the average volume of HA-SQSTM1 oligomerization. Average volume from 50 cells counted for each data point (bar). Statistical analysis via one-way ANOVA with Tukey's test, **P < 0.01, ***P < 0.001. (D) Confocal microscopy of HeLa cells co-transfected with GFP or GFP-TRIM44 and specific HA-SQSTM1 constructs (HA-SQSTM1, HA-SQSTM1K7R, HA-SQSTMQD69A, and HA-SQSTM1K7AD69A). Immunostaining with HA antibody performed post 48 h. Scale bars: 10 µm. (E) Graph of average volume of HA-SQSTM1 constructs oligomerization (HA-SQSTM1, HA-SQSTM1K7R, HA-SQSTM1D69A, and HA-SQSTM1K7AD69A). One-way ANOVA with Tukey's test for statistical analysis. ***P < 0.001. Black star (*) denotes GFP-TRIM44 transfected cells versus corresponding GFP transfected cells. Red star (*) denotes HA-SQSTM1 mutants’ transfected cells versus corresponding wild-type HA-SQSTM1 transfected cells. (F) In vivo deubiquitylation assay. 293T cells were co-transfected with HA-SQSTM1 constructs (HA-SQSTM1 and HA-SQSTM1K7AD69A), His-Ubiquitin and GFP-TRIM44 plasmids. Post 48 h, cells were treated with proteasome inhibitor MG132 (5 μM, 6 h), followed by immunoprecipitation with HA antibody. Anti-Ub antibody was used to immunoblot ubiquitinated HA-SQSTM1 and HA-SQSTM1K7AD69A. (G) HeLa cells were transfected with FLAG-HA-SQSTM1 or FLAG-HA-SQSTM1ΔPB1 mutant (lacking PB1 domain), in conjunction with either GFP or GFP-TRIM44. After 48 h, immunostaining was performed with HA (red) and DRAQ5 (blue). Scale bars represent 10 μm. (H) Graphic presentation of the average oligomerization volume of SQSTM1 (FLAG-HASQSTM1, FLAG-HA-SQSTM1ΔPB1). Each bar reflects the oligomerization volume of the indicated HA-SQSTM1 constructs in 50 cells. Statistical analysis was carried out using one-way ANOVA with Tukey's test, with **P < 0.01 and ***P < 0.001. (I) 293T cells were co-transfected with the indicated plasmids. After 48 h, cells were cross-linked with DSP (0.4 mg/ml, 4 °C, 2 h) and processed under either reducing (with β-ME) or nonreducing conditions (without β-ME). The lysates were then subjected to immunoblotting using the indicated antibodies.CC BY-NC-ND · PubMed Central
Figure 3 TRIM44 promotes SQSTM1 oligomerization in response to oxidative stresses. (A) Confocal microscopy of HeLa stable cells (TRIM44[OE-CON], or TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]) treated with Bafilomycin A1 (Baf A1, 400 nM, 4 h), or chloroquine (CQ, 50 μM, 4 h), or arsenic trioxide (As[III], 5 μM, 4 h), or hydrogen peroxide (H2O2, 3 mM, 10 min). Immunostaining with anti-SQSTM1 antibody was performed. Cells with more than two visible SQSTM1 aggregates were considered positive. n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.005 (Student's unpaired t test). (B) Immunofluorescence staining of HeLa cells co-transfected with indicated plasmids. After 48 h, cells were challenged with As[III] (5 μM, 24 h) and immunostained with anti-HA (magenta), anti-ubiquitin (red), and DRAQ5 (blue). Volume of oligomerized SQSTM1 was quantified (mean ± SD, n = 3, **P < 0.01, ***P < 0.005, one-way ANOVA with Tukey's test). (C) Immunofluorescence staining. HeLa cells co-transfected with indicated plasmids. After 24 h, cells were treated with or without NAC (0.2 mM, 24 h). Immunostaining with anti-HA antibody (red) and DRAQ5 (blue) was performed. Scale bars represent 20 μm. (D) Percentage of cells with SQSTM1 aggregates was quantified. Cells with more than two visible SQSTM1 aggregates were considered positive (n = 3, mean ± SD, **P < 0.01 (Student's unpaired t test). (E) Volume of oligomerized SQSTM1 was quantified. Student's unpaired t test was used for statistical analysis (***P < 0.001).
Figure 3 TRIM44 promotes SQSTM1 oligomerization in response to oxidative stresses. (A) Confocal microscopy of HeLa stable cells (TRIM44[OE-CON], or TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]) treated with Bafilomycin A1 (Baf A1, 400 nM, 4 h), or chloroquine (CQ, 50 μM, 4 h), or arsenic trioxide (As[III], 5 μM, 4 h), or hydrogen peroxide (H2O2, 3 mM, 10 min). Immunostaining with anti-SQSTM1 antibody was performed. Cells with more than two visible SQSTM1 aggregates were considered positive. n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.005 (Student's unpaired t test). (B) Immunofluorescence staining of HeLa cells co-transfected with indicated plasmids. After 48 h, cells were challenged with As[III] (5 μM, 24 h) and immunostained with anti-HA (magenta), anti-ubiquitin (red), and DRAQ5 (blue). Volume of oligomerized SQSTM1 was quantified (mean ± SD, n = 3, **P < 0.01, ***P < 0.005, one-way ANOVA with Tukey's test). (C) Immunofluorescence staining. HeLa cells co-transfected with indicated plasmids. After 24 h, cells were treated with or without NAC (0.2 mM, 24 h). Immunostaining with anti-HA antibody (red) and DRAQ5 (blue) was performed. Scale bars represent 20 μm. (D) Percentage of cells with SQSTM1 aggregates was quantified. Cells with more than two visible SQSTM1 aggregates were considered positive (n = 3, mean ± SD, **P < 0.01 (Student's unpaired t test). (E) Volume of oligomerized SQSTM1 was quantified. Student's unpaired t test was used for statistical analysis (***P < 0.001).CC BY-NC-ND · PubMed Central
Figure 4 Oligomerization of SQSTM1 is essential for its phosphorylation at S349. (A) HeLa cells stable expressing WT FLAG-HA-SQSTM1 (WT), FLAG-HA-SQSTM1 [C105/113A] (C105/113A) or FLAG-HA-SQSTM1[K102E] (K102E) were treated with As[III] (from 2.5 to 5 μM, 6 h), followed by cross-linking with DSP (0.4 mg/ml, 4 °C, 2 h). Cell lysates, prepared under reducing (with β-ME) or non-reducing condition (without β-ME), were subjected to immunoblotting for indicated antibodies. (B) 293T cells stably expressing WT FLAG-HA-SQSTM1, FLAG-HA SQSTM1[C105/113A] (C105/113A) or FLAG-HA-SQSTM1[K102E] (K102E) were treated with As[III] (from 2.5 to 5 μM, 6 h). Whole-cell lysates were examined for SQSTM1 p-S349, HA expression by immunoblotting (GAPDH was used as a loading control). (C) 293T cells stably co-expressing indicated SQSTM1 wild-type or SQSTM1 mutations with or without TRIM44 were treated with As[III] (5 μM) for 6 h. Immunoprecipation was performed with anti-HA antibody and immunoblotting for SQSTM1 p-S349 and HA. (D) Immunofluorescence staining of HeLa cells transfected with indicated HA-SQSTM1 constructs (HA-SQSTM1, and HA-SQSTM1S349A). After 48 h, cells were treated with or without As[III] (5 μM, 24 h). Immunostaining with anti-HA antibody was performed. Percentage of cells containing oligomerized SQSTM1 was analyzed. Cells with more than two visible SQSTM1 aggregates were considered positive. n = 3, mean ± SD. NS, non-significant (Student's unpaired t test). (E) 293T cells were transfected with HA-SQSTM1 constructs and expressed to MG132 (5 μM) for a duration of 6 h. The treated cells were subsequently cross-linked with DSP (0.4 mg/ml, 4 °C, 2 h) and prepared under either reducing conditions (with β-ME) or nonreducing condition (without β-ME). Cell lysates were subjected to immunoblotting for the indicated antibodies. Immunoprecipitation was executed using an anti-HA antibody and immunoblotting for SQSTM1 p-S349 and HA.
Figure 4 Oligomerization of SQSTM1 is essential for its phosphorylation at S349. (A) HeLa cells stable expressing WT FLAG-HA-SQSTM1 (WT), FLAG-HA-SQSTM1 [C105/113A] (C105/113A) or FLAG-HA-SQSTM1[K102E] (K102E) were treated with As[III] (from 2.5 to 5 μM, 6 h), followed by cross-linking with DSP (0.4 mg/ml, 4 °C, 2 h). Cell lysates, prepared under reducing (with β-ME) or non-reducing condition (without β-ME), were subjected to immunoblotting for indicated antibodies. (B) 293T cells stably expressing WT FLAG-HA-SQSTM1, FLAG-HA SQSTM1[C105/113A] (C105/113A) or FLAG-HA-SQSTM1[K102E] (K102E) were treated with As[III] (from 2.5 to 5 μM, 6 h). Whole-cell lysates were examined for SQSTM1 p-S349, HA expression by immunoblotting (GAPDH was used as a loading control). (C) 293T cells stably co-expressing indicated SQSTM1 wild-type or SQSTM1 mutations with or without TRIM44 were treated with As[III] (5 μM) for 6 h. Immunoprecipation was performed with anti-HA antibody and immunoblotting for SQSTM1 p-S349 and HA. (D) Immunofluorescence staining of HeLa cells transfected with indicated HA-SQSTM1 constructs (HA-SQSTM1, and HA-SQSTM1S349A). After 48 h, cells were treated with or without As[III] (5 μM, 24 h). Immunostaining with anti-HA antibody was performed. Percentage of cells containing oligomerized SQSTM1 was analyzed. Cells with more than two visible SQSTM1 aggregates were considered positive. n = 3, mean ± SD. NS, non-significant (Student's unpaired t test). (E) 293T cells were transfected with HA-SQSTM1 constructs and expressed to MG132 (5 μM) for a duration of 6 h. The treated cells were subsequently cross-linked with DSP (0.4 mg/ml, 4 °C, 2 h) and prepared under either reducing conditions (with β-ME) or nonreducing condition (without β-ME). Cell lysates were subjected to immunoblotting for the indicated antibodies. Immunoprecipitation was executed using an anti-HA antibody and immunoblotting for SQSTM1 p-S349 and HA.CC BY-NC-ND · PubMed Central
Figure 5 TRIM44 facilitates SQSTM1 S349 phosphorylation via PKA. (A) U266 TRIM44[OE-CON], TRIM44[OE] cells were exposed to inhibitors targeting CKI (CKI-7, 100 µM, 6 h), GSK3 (CHIR-99021, 2 µM, 24 h), CaMKII (KN-62, 1 µM, 24 h), and PKA (H89, 5 µM, 24 h), and As[III] (5 µM, 6 h) or left untreated. Whole-cell lysates were examined using anti-SQSTM1, anti-SQSTM1 p-S349. GAPDH served as a loading control. (B) Cell extracts from U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were lysed for measure PKA activity (PKA Kinase activity Kit, Abcam, ab139435). Measurements were made in triplicates, mean ± SD. Student's t test was used for statistical analysis *P < 0.05, **P < 0.01. (C) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without As[III] (5 μM, 6 h). Immunoprecipitation was subsequently performed with an anti-PKA substrate antibody and immunoblotting for indicated signals. (D) U266 TRIM44[KD-CON], TRIM44[KD] cells were treated with or without As[III] (5 μM, 24 h). Whole-cell lysates were analyzed via immunoblotting for SQSTM1 p-S349, SQSTM1, PKA Cα. (E) U266 TRIM44[OE-CON] and TRIM44[OE] cells were transfected with NC or PKA Cα siRNA. After 48 h, whole-cell lysates were analyzed via immunoblotting for SQSTM1 pS349 and SQSTM1, PKA Cα. (F) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without Baf A1 (100 nM, 6 h). Immunoprecipitation was subsequently performed with an anti-HA antibody and immunoblotting for indicated signals.
Figure 5 TRIM44 facilitates SQSTM1 S349 phosphorylation via PKA. (A) U266 TRIM44[OE-CON], TRIM44[OE] cells were exposed to inhibitors targeting CKI (CKI-7, 100 µM, 6 h), GSK3 (CHIR-99021, 2 µM, 24 h), CaMKII (KN-62, 1 µM, 24 h), and PKA (H89, 5 µM, 24 h), and As[III] (5 µM, 6 h) or left untreated. Whole-cell lysates were examined using anti-SQSTM1, anti-SQSTM1 p-S349. GAPDH served as a loading control. (B) Cell extracts from U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were lysed for measure PKA activity (PKA Kinase activity Kit, Abcam, ab139435). Measurements were made in triplicates, mean ± SD. Student's t test was used for statistical analysis *P < 0.05, **P < 0.01. (C) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without As[III] (5 μM, 6 h). Immunoprecipitation was subsequently performed with an anti-PKA substrate antibody and immunoblotting for indicated signals. (D) U266 TRIM44[KD-CON], TRIM44[KD] cells were treated with or without As[III] (5 μM, 24 h). Whole-cell lysates were analyzed via immunoblotting for SQSTM1 p-S349, SQSTM1, PKA Cα. (E) U266 TRIM44[OE-CON] and TRIM44[OE] cells were transfected with NC or PKA Cα siRNA. After 48 h, whole-cell lysates were analyzed via immunoblotting for SQSTM1 pS349 and SQSTM1, PKA Cα. (F) 293T cells stably co-expressing FLAG-HA-SQSTM1 with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with or without Baf A1 (100 nM, 6 h). Immunoprecipitation was subsequently performed with an anti-HA antibody and immunoblotting for indicated signals.CC BY-NC-ND · PubMed Central
Figure 6 TRIM44-mediated phosphorylation of SQSTM1 accelerates KEAP1 degradation. (A) Immunoblot analysis was conducted on whole-cell lysates from U266 TRIM44[OECON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells, examining for KEAP1. (B) HeLa TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were treated with cycloheximide (100 μg/ml) for specified durations. Whole-cell lysates were then analyzed via immunoblotting for KEAP1. GAPDH was utilized as a loading control. (C) Confocal microscopy of HeLa cells tri-transfected with FLAG-KEAP1, mCherry-SQSTM1 and GFP or GFP-TRIM44. After 24 h, cells were treated with or without As[III] for another 24 h. Immunostaining with anti-FLAG (red), and anti-SQSTM1 p-S349 (white) was performed. Expression of GFP-TRIM44 or GFP was shown as green, and mCherry-SQSTM1 was shown as magenta color. Scale bars represent 10 μm. (D) HeLa TRIM44[OE-CON] and TRIM44[OE] cells were treated with As[III] (5 μM) for 6 h. Whole-cell lysates were analyzed by immunoblotting. Non-Aggregates: NP-40-soluble; Aggregates: SDS-soluble, NP40 insoluble. Relative ratios of Non-Aggregates or Aggregates versus GAPDH are indicated. (E) HeLa cells were transfected with NC or TRIM44 siRNA. After 24 h, cells were then co-transfected with FLAG-KEAP1 and mCherry-SQSTM1 for another 24 h. Immunostaining with anti-FLAG antibody (green) and DRAQ5 (blue) were performed. Expression of mCherry-SQSTM1 was shown as magenta color. Scale bars represent 20 μm. Cells were also analyzed by immunoblotting for TRIM44, FLAG and mCherry. GAPDH was used as a loading control. Volume of KEAP1-positive oligomerized SQSTM1 was quantified. Student's unpaired t test was used for statistical analysis. **P < 0.01. (F) Immunofluorescence staining. HeLa TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON], TRIM44[KD] cells were tri-transfected with HA-SQSTM1, mCherry-LC3B, and FLAG-KEAP1. After 48 h, cells were immunostained with anti-HA (red), and anti-FLAG (blue). Expression of mCherry-LC3 was shown as green color. Scale bars, represent 10 μm. (G) 293T cells were co-transfected with FLAG-KEAP1 and mCherry-SQSTM1. After 48 h, the cells were treated with MG132 (5 μM, 6 h) and then treated with the crosslinking agent DSP (0.4 mg/ml, 4 °C) for 2 h. Subsequently, the cells were lysed in IP lysis buffer containing 1% SDS. The lysates were mixed with loading buffer containing β-ME for reducing condition or without β-ME (non-reducing condition). The samples were then subjected to SDS-PAGE and immunoblotting for SQSTM1 and KEAP1.
Figure 6 TRIM44-mediated phosphorylation of SQSTM1 accelerates KEAP1 degradation. (A) Immunoblot analysis was conducted on whole-cell lysates from U266 TRIM44[OECON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells, examining for KEAP1. (B) HeLa TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were treated with cycloheximide (100 μg/ml) for specified durations. Whole-cell lysates were then analyzed via immunoblotting for KEAP1. GAPDH was utilized as a loading control. (C) Confocal microscopy of HeLa cells tri-transfected with FLAG-KEAP1, mCherry-SQSTM1 and GFP or GFP-TRIM44. After 24 h, cells were treated with or without As[III] for another 24 h. Immunostaining with anti-FLAG (red), and anti-SQSTM1 p-S349 (white) was performed. Expression of GFP-TRIM44 or GFP was shown as green, and mCherry-SQSTM1 was shown as magenta color. Scale bars represent 10 μm. (D) HeLa TRIM44[OE-CON] and TRIM44[OE] cells were treated with As[III] (5 μM) for 6 h. Whole-cell lysates were analyzed by immunoblotting. Non-Aggregates: NP-40-soluble; Aggregates: SDS-soluble, NP40 insoluble. Relative ratios of Non-Aggregates or Aggregates versus GAPDH are indicated. (E) HeLa cells were transfected with NC or TRIM44 siRNA. After 24 h, cells were then co-transfected with FLAG-KEAP1 and mCherry-SQSTM1 for another 24 h. Immunostaining with anti-FLAG antibody (green) and DRAQ5 (blue) were performed. Expression of mCherry-SQSTM1 was shown as magenta color. Scale bars represent 20 μm. Cells were also analyzed by immunoblotting for TRIM44, FLAG and mCherry. GAPDH was used as a loading control. Volume of KEAP1-positive oligomerized SQSTM1 was quantified. Student's unpaired t test was used for statistical analysis. **P < 0.01. (F) Immunofluorescence staining. HeLa TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON], TRIM44[KD] cells were tri-transfected with HA-SQSTM1, mCherry-LC3B, and FLAG-KEAP1. After 48 h, cells were immunostained with anti-HA (red), and anti-FLAG (blue). Expression of mCherry-LC3 was shown as green color. Scale bars, represent 10 μm. (G) 293T cells were co-transfected with FLAG-KEAP1 and mCherry-SQSTM1. After 48 h, the cells were treated with MG132 (5 μM, 6 h) and then treated with the crosslinking agent DSP (0.4 mg/ml, 4 °C) for 2 h. Subsequently, the cells were lysed in IP lysis buffer containing 1% SDS. The lysates were mixed with loading buffer containing β-ME for reducing condition or without β-ME (non-reducing condition). The samples were then subjected to SDS-PAGE and immunoblotting for SQSTM1 and KEAP1.CC BY-NC-ND · PubMed Central
Figure 7 TRIM44 regulates the SQSTM1‐KEAP1‐NFE2L2 system. (A) U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were treated with or without As[III] (5 μM, 6 h). Nuclear fractions and cytoplasmic fractions were prepared and then subjected to immunoblot analysis with indicated antibodies. Relative levels of NFE2L2 in nuclear and KEAP1 in cytoplasm were quantified. (B) Quantitation of messenger RNA (mRNA) levels of NFE2L2 targets in U266 stable cells. Cells were challenged with or without As[III] (5 μM, 24 h) and then subjected to qRT-PCR analysis with indicated genes. The experiments were performed three times. Data are mean ± SD. Statistical analysis was carried out using Student’s t test, with *P < 0.05, **P < 0.01 and ***P < 0.001. (C) Immunoprecipitation assays. 293T (TRIM44[OE-CON], TRIM44[OE], TRIM44[KDCON], and TRIM44[KD]) cells were tri-transfected with indicated plasmids. After 48 h, cells were subjected to immunoprecipitation with anti-GFP and anti-HA. Immunoblotting was performed with indicated antibodies. Data are representative of three independent experiments. (D) Immunoprecipitation assays. 293T cells were co-transfected with indicated plasmids. After 48 h, cells were treated with MG132 (5 μM) for 6 h and then subjected to immunoprecipitation with anti-HA. Immunoblotting was performed with indicated antibodies. Data are representative of three independent experiments. (E) Immunoblot analysis. 293T cells stably co-expressing FLAG-HA-SQSTM1 wild-type (WT) or its mutation (C105/113A) with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with As[III] (5 μM, 6 h). Total cell lysates and nuclear fractions were prepared and analyzed by immunoblotting for indicated antibodies.
Figure 7 TRIM44 regulates the SQSTM1‐KEAP1‐NFE2L2 system. (A) U266 TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] cells were treated with or without As[III] (5 μM, 6 h). Nuclear fractions and cytoplasmic fractions were prepared and then subjected to immunoblot analysis with indicated antibodies. Relative levels of NFE2L2 in nuclear and KEAP1 in cytoplasm were quantified. (B) Quantitation of messenger RNA (mRNA) levels of NFE2L2 targets in U266 stable cells. Cells were challenged with or without As[III] (5 μM, 24 h) and then subjected to qRT-PCR analysis with indicated genes. The experiments were performed three times. Data are mean ± SD. Statistical analysis was carried out using Student’s t test, with *P < 0.05, **P < 0.01 and ***P < 0.001. (C) Immunoprecipitation assays. 293T (TRIM44[OE-CON], TRIM44[OE], TRIM44[KDCON], and TRIM44[KD]) cells were tri-transfected with indicated plasmids. After 48 h, cells were subjected to immunoprecipitation with anti-GFP and anti-HA. Immunoblotting was performed with indicated antibodies. Data are representative of three independent experiments. (D) Immunoprecipitation assays. 293T cells were co-transfected with indicated plasmids. After 48 h, cells were treated with MG132 (5 μM) for 6 h and then subjected to immunoprecipitation with anti-HA. Immunoblotting was performed with indicated antibodies. Data are representative of three independent experiments. (E) Immunoblot analysis. 293T cells stably co-expressing FLAG-HA-SQSTM1 wild-type (WT) or its mutation (C105/113A) with TRIM44 (TRIM44[OE]) or without TRIM44 (TRIM44[OE-CON) were treated with As[III] (5 μM, 6 h). Total cell lysates and nuclear fractions were prepared and analyzed by immunoblotting for indicated antibodies.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Wang, Y., Lyu, L., Vu, T., & McCarty, N.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2024-08-16
Volume
14
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-024-67832-x
PMC
PMC11329658
Cited by
8
References
2
Language
en
Licence
Funders
National Institute of Health, United States (CA181319)

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