Journal: bioRxiv

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1101/2023.03.16.532226

Figure Lengend Snippet: ( A ) Western blot of Triton-soluble lysate (left lanes) and YFP-PKCβII immunoprecipitated from COS7 cells using GFP-Trap ® Agarose. Cells were pre-treated with 20 μM MG-132 for 3 hours followed by 30 min of 200 nM PDBu treatment prior to lysis. Blots were probed with indicated antibodies. ( B ) Quantification of PDBu-induced ubiquitination of immunoprecipitated PKCβII. Relative ubiquitination was determined (Ubiquitin / PKC) for immunoprecipitated samples and each condition was normalized to DMSO-treated control (1.0) to determine fold-increase in ubiquitination after PDBu stimulation. Data represent mean ± SEM from four independent experiments. ns = not significant, **** P < 0.0001 by two-way ANOVA and Šídák’s multiple comparisons test.

Article Snippet: Antibodies against GFP (catalog no. 2555S), Vinculin (catalog no. 4650S), Ubiquitin (catalog no. 3933S), and GAPDH (14C10, catalog no. 2118) were from Cell Signaling Technologies and used at 1:1000 dilution.

Techniques: Western Blot, Immunoprecipitation, Lysis

Journal: The Journal of Clinical Investigation

Article Title: Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models

doi: 10.1172/JCI159638

Figure Lengend Snippet: ( A ) IP of anti-HA with cell lysate from 293T cells transfected with HA-Plek2, followed by Western blot assays of indicated proteins. ( B ) Western blotting assays of indicated proteins from Cos-7 cells transfected with HA-Plek2. ( C ) Quantitative PCR analysis of AKT from Cos-7 cells transfected with HA-Plek2. ( D ) Western blotting assays of indicated proteins from HEL cells transfected with Flag-Plek2. ( E ) GST pull-down assay using GST or GST-Plek2 with a recombinant Akt. Akt was detected by a Western blotting assay. GST and GST-Plek2 were revealed using Coomassie stain. ( F ) Same as E , except GST-Plek2 DEP was used. ( G ) GST pull-down assay of GST or GST-Plek2 incubated with cell lysis from 293T cells. Indicated proteins were detected by Western blotting. ( H ) Bead-conjugated streptavidin pull-down of 293T cells transfected with BirA-Plek2 and incubated with biotin (50 μM). A Western blotting assay of the indicated proteins was performed after SDS-PAGE. ( I ) Western blotting analyses of the indicated proteins in 293T cells transfected with GFP fusion Hsp72 and HA-GST-Akt. ( J ) IP of Flag tag with cell lysate from 293T cells transfected with Flag-Plek2 followed by a Western blotting assay of indicated proteins.

Article Snippet: For Western blotting, the following antibodies were used: anti-HSC70 (catalog sc-7298) (Santa Cruz Biotechnology Inc.); anti-Pten (catalog 9559), anti-total AKT (catalog 4691), anti-phospho AKT (catalog 4060), anti-total S6 (catalog 2217), anti-phospho S6 (catalog 4858), anti-phospho GSK3b(catalog 5558), anti-mTOR (catalog 2972), anti-GFP (catalog 2956), anti-GST (catalog 2622), anti-Flag (catalog 8146), and anti-HA (catalog 3724) (Cell Signaling Technology); anti-Plek2 (catalog 11685-1-AP) and anti-PDK2 (catalog 15647-1-AP) (Proteintech); anti-Hsp72 (catalog PA5-34772) (Invitrogen); and HRP linked anti-GST antibody (catalog MA4-004-HRP) (Thermo Fisher Scientific).

Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Pull Down Assay, Recombinant, Staining, Incubation, Lysis, SDS Page, FLAG-tag

Journal: Nature Communications

Article Title: Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish

doi: 10.1038/s41467-023-37026-6

Figure Lengend Snippet: a Strategy for the generation of Ara and Arb KI medaka strains. We designed a gRNA targeting ara intron 8 and arb intron 7 (the last intron). In the donor plasmids, we cloned a genomic fragment beginning from the end of exon 8 of ara and exon 7 of arb and ending just before the stop codon in the last exon, exon 9 of ara , and exon 8 (shown as black closed boxes with ‘E8’) of arb , where the 3xFLAG sequences (shown as blue boxes) and a P2A (2A peptide from porcine teschovirus-1)-mClover3 cassette (shown as green boxes) was placed in the frame. Thus, endogenous Ara and Arb were expressed as FLAG fusion proteins. Both AR-FLAG and P2A-mClover3 were expected to be expressed under the control of the endogenous Ar promoter. To generate KI medaka, sgRNA (for genome digestion in the final intron), donor plasmid, and Cas9 mRNA were co-injected into one-cell-stage medaka embryos. After injection, concurrent cleavage of the targeted genomic locus and the donor plasmid resulted in the integration of donor plasmid DNA containing 3xFLAG-T2A-mClover3 by non-homologous end joining (NHEJ). The scheme shows the forward integration of 3xFLAG-T2A-mClover3. b Expression of mClover3 in adult males of the two knock-in medaka strains, ara FLAG-2A-mClover3 (Ara-KI) and arb FLAG-2A-mClover3 (Arb-KI). White, grey, and red arrows indicate the regions adjacent to pectoral, dorsal, and anal fins, respectively (Ara-KI). White, grey, and red arrows indicate the pectoral, dorsal, and anal fins, respectively (Arb-KI). c Immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in longitudinal sections of papillary processes of the anal fin (6 μm thickness). The merged images represent red fluorescence for immunostaining of FLAG (anti-DDDDK-tag mouse mAb monoclonal antibody), green fluorescence for immunostaining of mClover3 (anti-GFP D5.1XP rabbit mAb monoclonal antibody), and blue fluorescence for nuclear staining by DAPI. The medaka Arb-FLAG, but not Ara-FLAG, translocated into the nuclei of cells located in the distal tip of a bone nodule of papillary processes (marked by white arrows). d Representative micrographs of Masson/trichrome staining and immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in adjacent sections of the urogenital region (8 μm thickness). Nuclear localisation of Ara-FLAG and Arb-FLAG was observed in the medulla ventral to the sperm duct, where ar DKO exhibited hyperplasia. e, f Representative micrographs showing immunohistochemical detection of FLAG-tagged endogenous Ara and Arb, and DAPI counterstaining in the brain (12 μm thickness). f shows a higher magnification of the POA. Nuclear localisation of both Ara-FLAG and Arb-FLAG was observed in the POA. n = 6 for Ara-KI and Arb-KI males, respectively.

Article Snippet: Briefly, after antigen retrieval by incubating slides in 0.1 mM citrate buffer (pH 6.0) in an autoclave (121 °C) for 1 min, the sections were blocked for 1 h using 1xPBS containing 0.1% Tween 20, 2% BSA, and 2% foetal bovine serum, and incubated with anti-DDDDK-tag (FLAG) mouse mAb monoclonal antibody (M185-3L, MBL, Nagoya, Japan, 1:300 dilution) and anti-GFP D5.1XP rabbit mAb monoclonal antibody having cross-reactivity to the mClover3 (#2956, Cell Signaling, Danvers, MA, USA, 1:300 dilution) overnight at 4 °C.

Techniques: Clone Assay, Plasmid Preparation, Injection, Non-Homologous End Joining, Expressing, Knock-In, Immunohistochemical staining, Fluorescence, Immunostaining, Staining

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Chlamydomonas expressing KAP-GFP. Indicated are the measured areas of KAP-GFP localization: the two basal bodies and cilia. Scale bar is 5 μm. (B) Super plot quantification of basal body fluorescence in (A) after a 2-h treatment with BCI at the indicated concentrations. Error bars are the mean with the 95% confidence interval for the averages from the trials. P = 0.5126, which was determined by an ordinary one-way ANOVA. (C) Ciliary length plotted against ciliary fluorescence for cells in DMSO (orange circles) or 30 μM BCI (purple) after a 2-h treatment (n = 40, N = 1). Simple linear regression was used to generate linear regression lines and comparisons. For DMSO, y = 0.1171x + 0.4305 and r 2 = 0.05676. For BCI, y = 0.1174x + 0.4051 and r 2 = 0.2636. Comparing slopes, F = 4.048e-006 (1, 76) and P = 0.9984. Comparing intercepts, F = 0.005429 (1, 77) and P = 0.9415. (D) Comparison of total ciliary fluorescence shown in (C). P -values were determined using a t test. (E) Comparisons of ciliary fluorescence in DMSO or BCI per micrometer of cilia in (C). P -values were determined using a t test. (F) Example kymographs collected from total internal reflection fluorescent microscopy of KAP-GFP movement in cilia in cells treated with DMSO (top) or 30 μM BCI (bottom) for 2 h. Vertical scale bars are 4 μm. Horizontal scale bars are 2 s. (G, H, I) KAP-GFP dynamics quantified from the kymographs (n = 20, N = 1). Error bars are the mean with the 95% confidence interval (n = 20, N = 1). P -values were calculated from a two-tailed unpaired t test for pairwise comparisons. (G) Frequency of KAP-GFP trains measured as the total amount of trains counted over the total amount of time the kymograph was collected. For DMSO, n = 40 (1 and 2 h). For BCI, n = 30 (1 h) and 34 (2 h). (H) Velocity of KAP-GFP trains measured as the distance traveled in μm over time in seconds. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). (I) Relative injection size of KAP-GFP trains measured as the relative total fluorescent intensity of each train relative to the maximum measurement. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). P -values were determined using a t test.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Two Tailed Test, Injection

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Immunofluorescent images of KAP-GFP cells during regeneration in either DMSO or 30 μM BCI. Scale bars are 5 μm. Red arrows point to basal bodies, which are the object of quantification in (D). (B) Western blot for KAP-GFP expression in regenerating cells in either DMSO (D) or 30 μM BCI (B). Total protein was measured with amido black. (C) Quantification of (B). Error bars are the mean with SD for three independent experiments. P -values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (D) Quantification of KAP-GFP fluorescence at the basal bodies in (A). Error bars are the mean with the 95% confidence interval for the averages from three independent trials (n = 30, N = 3). The P -value was calculated using an unpaired t test with Welch’s correction.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Western blot of KAP-GFP expression compared with total protein shown with amido black. Cells were treated for 2 h with either 0.5% DMSO, 20 μM BCI, 50 μM MG132, or both 50 μM MG132 and 20 μM BCI. (A, B) Quantification of (A). Error bars are SD of the mean (N = 3). The P -value was determined by an unpaired t test between BCI and BCI + MG132.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: BCI inhibits phosphate removal from MAPK. This ultimately alters or inhibits ciliogenesis, KAP-GFP dynamics in cilia, KAP-GFP protein synthesis, NPHP4 protein localization at the transition zone, membrane trafficking, and microtubule organization.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques:

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: Tools and reagents.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Staining, Mutagenesis

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: Tools and reagents.

Article Snippet: GFP (D5.1) rabbit mAb , Cell Signaling Technology , 2956S.

Techniques: Staining, Mutagenesis

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Chlamydomonas expressing KAP-GFP. Indicated are the measured areas of KAP-GFP localization: the two basal bodies and cilia. Scale bar is 5 μm. (B) Super plot quantification of basal body fluorescence in (A) after a 2-h treatment with BCI at the indicated concentrations. Error bars are the mean with the 95% confidence interval for the averages from the trials. P = 0.5126, which was determined by an ordinary one-way ANOVA. (C) Ciliary length plotted against ciliary fluorescence for cells in DMSO (orange circles) or 30 μM BCI (purple) after a 2-h treatment (n = 40, N = 1). Simple linear regression was used to generate linear regression lines and comparisons. For DMSO, y = 0.1171x + 0.4305 and r 2 = 0.05676. For BCI, y = 0.1174x + 0.4051 and r 2 = 0.2636. Comparing slopes, F = 4.048e-006 (1, 76) and P = 0.9984. Comparing intercepts, F = 0.005429 (1, 77) and P = 0.9415. (D) Comparison of total ciliary fluorescence shown in (C). P -values were determined using a t test. (E) Comparisons of ciliary fluorescence in DMSO or BCI per micrometer of cilia in (C). P -values were determined using a t test. (F) Example kymographs collected from total internal reflection fluorescent microscopy of KAP-GFP movement in cilia in cells treated with DMSO (top) or 30 μM BCI (bottom) for 2 h. Vertical scale bars are 4 μm. Horizontal scale bars are 2 s. (G, H, I) KAP-GFP dynamics quantified from the kymographs (n = 20, N = 1). Error bars are the mean with the 95% confidence interval (n = 20, N = 1). P -values were calculated from a two-tailed unpaired t test for pairwise comparisons. (G) Frequency of KAP-GFP trains measured as the total amount of trains counted over the total amount of time the kymograph was collected. For DMSO, n = 40 (1 and 2 h). For BCI, n = 30 (1 h) and 34 (2 h). (H) Velocity of KAP-GFP trains measured as the distance traveled in μm over time in seconds. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). (I) Relative injection size of KAP-GFP trains measured as the relative total fluorescent intensity of each train relative to the maximum measurement. For DMSO, n = 100 (1 and 2 h). For BCI, n = 93 (1 h) and 88 (2 h). P -values were determined using a t test.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Two Tailed Test, Injection

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Immunofluorescent images of KAP-GFP cells during regeneration in either DMSO or 30 μM BCI. Scale bars are 5 μm. Red arrows point to basal bodies, which are the object of quantification in (D). (B) Western blot for KAP-GFP expression in regenerating cells in either DMSO (D) or 30 μM BCI (B). Total protein was measured with amido black. (C) Quantification of (B). Error bars are the mean with SD for three independent experiments. P -values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (D) Quantification of KAP-GFP fluorescence at the basal bodies in (A). Error bars are the mean with the 95% confidence interval for the averages from three independent trials (n = 30, N = 3). The P -value was calculated using an unpaired t test with Welch’s correction.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: (A) Western blot of KAP-GFP expression compared with total protein shown with amido black. Cells were treated for 2 h with either 0.5% DMSO, 20 μM BCI, 50 μM MG132, or both 50 μM MG132 and 20 μM BCI. (A, B) Quantification of (A). Error bars are SD of the mean (N = 3). The P -value was determined by an unpaired t test between BCI and BCI + MG132.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: BCI inhibits phosphate removal from MAPK. This ultimately alters or inhibits ciliogenesis, KAP-GFP dynamics in cilia, KAP-GFP protein synthesis, NPHP4 protein localization at the transition zone, membrane trafficking, and microtubule organization.

Article Snippet: To probe for KAP-GFP, blots were incubated with 5% milk and then probed with GFP antibody (1956S; CST) diluted at 1:1,000 in 1% milk + 1% BSA overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Dual role of CASP8AP2/FLASH in regulating epithelial-to-mesenchymal (EMT) plasticity

doi: 10.1101/2023.03.12.532282

Figure Lengend Snippet: (A) SNAIL EMT-TF upregulation in FLASH KD cells was determined by qPCR at mRNA level and Western blot analysis at protein level. (B) Hybrid EMT phenotype in FLASH-depleted cells as evidenced by distinct expression patterns of ECAD, SNAIL and ZEB1 (left panel). Overexpression of either ZEB1 (ZEB1-Myc), FLASH (FLASH-GFP) or SNAIL (SNAIL-Flag) inhibits E-cadherin expression (ECAD) in cancer cells (right panel). (C) Scatter plots of SNAIL and SLUG levels and the Mesenchymal score in TGFβ-treated and untreated FLASH, NPAT and SLBT-depleted cells. Spearman’s correlation coefficient (ρ) and corresponding p-value ( p ) are reported. Two independent experiments were used for the analysis. (D) Heat map analysis of mesenchymal genes altered in FLASH, NPAT and SLBT-depleted cell under no treatment (left panel) and TGFβ-treated conditions (right panel). (E) Mesenchymal markers ACTA2 and POSTN expression was determined by qPCR at mRNA level in WT, SNAIL KO and SLUG KO in control cells and FLASH-depleted cells. The significance of differences was confirmed by Student t test for silencing efficiency (*, p < 0.05; **, p < 0.01).

Article Snippet: The following antibodies were all purchased from Cell Signaling: FLASH (D3T8Q), ZEB1 (E2G6Y), SNAIL (C15D3), SLUG (C19G7), Claudin-6 (E7U20), LYRIC (D5Y8R), RUFY3 (61460S), PJA2 (40180S), GFP (D5.1), Myc (9B11), Histone H3 (D1H2) and Histone H4 (D2X4V).

Techniques: Western Blot, Expressing, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes

doi: 10.3389/fphar.2023.1094584

Figure Lengend Snippet: Celastrol induced 3T3-L1 preadipocytes apoptosis through inhibition of autophagy (A) , Real-time qPCR of genes were studied in 3T3-L1 preadipocytes after 0, 2 and 4 μM celastrol treatment for 16 h ( n = 6) (B) , Western blotting of P62 and LC3 I/II were developed in 3T3-L1 preadipocytes after 0, 1 and 2 μM celastrol treatment for 24 h ( n = 3) (C) , Western blotting of P62 and LC3 I/II were developed in preadipocytes after 2 μM celastrol treatment for 24 h with or without 15 μM rapamycin, 40 μM chloroquine or 200 nM bafilomycin A1 ( n = 3) (D) , Autophagosome degradation was observed with RFP-GFP-LC3 adeno-associated virus, after 24 h treatment of 1 μM celastrol, 40 μM chloroquine or 15 μM rapamycin, respectively ( n = 7). Red fluorescence represented normal autophagosome degradation, while yellow represented halt of degradation (E–F) , 3T3-L1 preadipocytes were treated with 24 h of celastrol, 15 μM rapamycin, 40 μM chloroquine, 200 nM bafilomycin A1, celastrol + 15 μM rapamycin, celastrol + 40 μM chloroquine or celastrol + 200 nM bafilomycin A1, respectively, and subjected to flow cytometry analysis (E) ( n = 7) and Western blotting of cleaved-Caspase3 (F) ( n = 3). Protein expression was calculated relative to β -actin and depicted at the top of each blot. Error bars represent SEM; * p < 0.05; ** p < 0.01; *** p < 0.001. Veh, vehicle; Cela, celastrol; 1C, 1 μM celastrol; 2C, 2 μM celastrol; BafA1, bafilomycin A1; CQ, chloroquine; Rapa, rapamycin; Cas 3, Caspase 3.

Article Snippet: Preadipocytes infected with RFP-GFP-LC3 adeno-associated virus were treated with 1 μM celastrol, 40 μM chloroquine or 200 nM bafilomycin A1 for 24 h. Cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, then subjected to primary antibody LAMP1 (Cell Signaling Technology, United States, Cat #9091, RRID:AB_2687579, 1:100 dilution) overnight, followed by incubation with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (Abcam, United States, Cat # ab150079, RRID:AB_2722623, 1:500 dilution).

Techniques: Inhibition, Western Blot, Fluorescence, Flow Cytometry, Expressing

Journal: Frontiers in Pharmacology

Article Title: Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes

doi: 10.3389/fphar.2023.1094584

Figure Lengend Snippet: Celastrol inhibited the fusion of autophagosome and lysosome (A–B) , 3T3-L1 preadipocytes were treated with 24 h of 1 μM celastrol, 40 μM chloroquine or 200 nM bafilomycin A1, and subjected to LysoTracker red staining (A) ( n = 3) and immunofluorescence staining of LAMP1 and LC3 (B) . The representative images were shown on the left, and the Pearson correlation coefficient (PCC) for the colocalization of RFP-LC3 and Alexa Fluor 647-LAMP1 were presented on the upper right ( n = 3) (C) , 3T3-L1 preadipocytes were treated with 1 and 2 μM celastrol for 24 h and subjected to Western blotting of LAMP1 ( n = 3) (D) , 3T3-L1 preadipocytes were treated with 1, 2 μM celastrol and 40 μM chloroquine for 12 h and subjected to electron microscopy ( n = 5) (E) , 3T3-L1 adipocytes were treated with 1 and 2 μM celastrol for 24 h and subjected to Western blotting of P62 and LC3 I/II ( n = 3). Protein expression was calculated relative to β -actin or GAPDH and depicted at the top of each blot. Error bars represent SEM. Veh, vehicle; Cela, celastrol; CQ, chloroquine; BafA1, bafilomycin A1; AP, autophagosome.

Article Snippet: Preadipocytes infected with RFP-GFP-LC3 adeno-associated virus were treated with 1 μM celastrol, 40 μM chloroquine or 200 nM bafilomycin A1 for 24 h. Cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, then subjected to primary antibody LAMP1 (Cell Signaling Technology, United States, Cat #9091, RRID:AB_2687579, 1:100 dilution) overnight, followed by incubation with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (Abcam, United States, Cat # ab150079, RRID:AB_2722623, 1:500 dilution).

Techniques: Staining, Immunofluorescence, Western Blot, Electron Microscopy, Expressing