Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 is required for aggregates deaggregation and clearance in MM cells. (a, b) U266 cells (TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]) were treated withor without bortezomib (5 nM) for 24 h to induce aggregates formation. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (a). Scale bars: 10 μm. The status of aggregates after MG132 treatment was quantified in the histogram (b). *, P < 0.05; **, P < 0.01. (c) Quantification of the total volume of aggregates in U266 cells (TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]). The volume of aggregates in 100 cells was counted for each data point shown. (d) Western blot analysis of non-aggregates and aggregates fractions of U266 cells (TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]) treated with bortezomib (5 nM, 24 h) and probe with indicated antibodies. Non-aggregates and aggregates protein were fractionated using NP40 lysis buffer. After centrifugation, the supernatant was used as non-aggregates fraction and the pellet was extracted in 1% SDS to obtain aggregates protein fraction. (e-g) RPMI-TRIM44[Tet-on] cells were treated with or without DOX (1 µg/mL, 24 h) together with bortezomib (10 nM, 24 h) treatment to induce aggregates formation. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin. Scale bars: 10 μm. The protein level of TRIM44 were assayed by western blot (e). The status of aggregates after bortezomib treatment was quantified in the histogram (g). *, P < 0.05. (h) Quantification of the total volume of aggregates in RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL, 24 h) together with bortezomib (10 nM, 24 h) treatment to induce aggregates formation. The volume of aggregates in 100 cells was counted for each data point shown. *, P < 0.05.

Article Snippet: RPMI8226 and U266 cells were cultured in RPMI medium (GE Healthcare Life Sciences, SH30255.01) with 10% FBS.

Techniques: Staining, Western Blot, Lysis, Centrifugation

Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 leads to autophagy activities in MM cells. (a, b) The protein levels of LC3B and SQSTM1 of TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD] MM cells (U266 and RPMI) were assayed by western blots. (c, d) U266 cells (TRIM44[OE-CON], TRIM44[OE], TRIM44[KD-CON] and TRIM44[KD]) were treated with or without CQ (10 μM, 24 h). The protein levels of LC3B were assayed by western blots. (e) TRIM44[OE-CON] and TRIM44[OE] MM cells (U266) were treated with or without chloroquine CQ (10 μM, 24 h). And then cells were fixed and assayed for the appearance of autophagosomes by confocal microscopy. LC3B puncta were analyzed by LC3B-II antibody (magnification is 10 × 60 oil). Scale bars: 20 μm. Quantification of autophagosome formation. Cells with eight or more LC3B puncta were considered to have accumulated autophagosomes. Data are presented as mean % LC3B-positive cells ± SEM in three independent experiments. In each treatment at least 100 cells were analyzed (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (f) TRIM44[KD-CON] and TRIM44[KD] MM cells (U266) were treated with or without CQ (10 μM, 24 h). And then cells were fixed and assayed for the appearance of autophagosomes by confocal microscopy. LC3B puncta were analyzed by LC3B-II antibody (magnification is 10 × 60 oil). Scale bars: 20 μm. Quantification of autophagosome formation. Cells with eight or more LC3B puncta were considered to have accumulated autophagosomes. Data are presented as mean % LC3B-positive cells ± SEM in three independent experiments. In each treatment at least 100 cells were analyzed (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (g) RPMI-TRIM44[Tet-on] cells were treated with or without DOX (1 µg/mL) together CQ (10 μM, 24 h). The protein levels of LC3B were assayed by western blots. (h) RPMI-TRIM44[Tet-on] cells were treated with or without DOX (1 µg/mL) together with CQ (10 μM, 24 h). And then cells were fixed and assayed for the appearance of autophagosomes by confocal microscopy. LC3B puncta were analyzed by LC3B-II antibody (magnification is 10 × 60 oil). Scale bars: 20 μm. Quantification of autophagosome formation. Cells with eight or more LC3B puncta were considered to have accumulated autophagosomes. Data are presented as mean % LC3B-positive cells ± SEM in three independent experiments. In each treatment at least 100 cells were analyzed (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: RPMI8226 and U266 cells were cultured in RPMI medium (GE Healthcare Life Sciences, SH30255.01) with 10% FBS.

Techniques: Western Blot, Confocal Microscopy

Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 promtoes aggregates deaggregation and clearance via autophagy. (a) Cells were treated with MG132 and immunostained with antibodies to ubiquitin (red) and LC3B-II (green). Arrows indicate ubiquitin-positive aggregates that colocalize with LC3B-positive autophagosomes. Scale bars: 10 μm. (b) Confocal images of TRIM44[OE-CON] and TRIM44[OE] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA (10 mM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (c) Confocal images of TRIM44[KD-CON] and TRIM44[KD] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM). Arrows indicate cells with remaining aggregates. Scale bars: 10 µm. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (d, e) Confocal images of WT or ATG5 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (d). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (e). (f, g) Confocal images of WT or BECN1 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells were treated with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (f). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of BECN1 and TRIM44 were assayed by western blot (g). (h, i) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. The status of aggregates after MG132 treatment was quantified in the histogram. Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (h). (j) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) together with MG132 (5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm.

Article Snippet: RPMI8226 and U266 cells were cultured in RPMI medium (GE Healthcare Life Sciences, SH30255.01) with 10% FBS.

Techniques: Transfection, Staining, Western Blot