Journal: Disease Models & Mechanisms

Article Title: Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy

doi: 10.1242/dmm.049427

Figure Lengend Snippet: The stable myh9b heterozygous mutant deteriorated bag3 cardiomyopathy at adult stage. (A) Schematic of the predicted MMEJ-inducing myh9b genetic lesion of 8-nucleotides generated by injection of an sgRNA targeting sequences within the 12th exon. Dashed lines indicate an 8-nucleotide-long deletion. (B) Chromographs illustrating the sequences of the wild-type myh9b and the mutant alleles with the predicted 8-nucleotide-long deletion in F0 and F1 fish. (C) Quantification of cardiac function. Ejection fraction (EF) (in %) measured by using echocardiography in bag3 e2/e2 ;myh9b e12/+ double-mutant fish compared to single-mutant and WT control fish at 6 months; n= 7-11, one-way ANOVA. (D) Left panel: Representative images of isolated hearts from fish as indicated. Right panel: Quantification of ventricular surface area (VSA) normalized to the body weight (BW) of fish at 6 months; n= 7, one-way ANOVA. (E) Western blotting (top) and quantification LC3 II protein levels (bottom) of hearts from bag3 e2/e2 ; myh9b e12/+ double-mutant fish, bag3 e2/e2 , myh9b e12/+ or WT control fish at 6 months. Levels of Gapdh were used as control. n= 4, one-way ANOVA. (F) TEM images of bag3 e2/e2 ; myh9b e12/+ double-mutant fish hearts, bag3 e2/e2 , myh9b e12/+ and WT controls at 6 months. Asterisks indicate Z -disc aggregation. Scale bar: 2 µM (D). * P <0.05. ns, not significant.

Article Snippet: The following primary antibodies were used: anti-GAPDH (1:5000; Santa Cruz Biotechnology Inc., catalog #sc-25778), anti-mTOR (1:3000; Cell Signaling Technology, catalog #2983), anti-ubiquitin (1:1000; ThermoFisher Scientific, catalog #PA5-17067) and anti-LC3 (1:3000; Cell Signaling Technology, catalog #12741).

Techniques: Mutagenesis, Generated, Injection, Isolation, Western Blot

Journal: The Journal of Cell Biology

Article Title: The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA

doi: 10.1083/jcb.202201027

Figure Lengend Snippet: Arsenic-induced degradation of the PML/RARA oncoprotein requires p97. (A) NB4 cells were treated with the indicated concentrations of CB-5083 and exposed to arsenic (As) for the indicated time (h). Cell lysates were analyzed by Western blotting using an antibody to PML, RARA, p97, and NPLOC4. (B) NB4 cells were either untreated (−), treated with CB-5083 (CB), bafilomycin (Baf), or bortezomib (Bor). Response to arsenic (6 h) was investigated for all conditions. Cell lysates were analyzed by Western blotting using antibodies to PML, RARA, LC3, or ubiquitin. Molecular weight markers are indicated in kD. Source data are available for this figure: .

Article Snippet: Antibodies: Chicken anti-PML (homemade), mouse anti-Lamin A/C (SAB4200236; Sigma-Aldrich), sheep anti-SUMO-1 (homemade), rabbit anti-SUMO-2 (4971S; Cell signaling), rabbit FK2 ubiquitin (Enzo BML-PW8810-0500), rabbit anti-p97 (ab109240; Abcam), sheep anti-UFD1 (MRC Dundee), sheep anti-NPLOC4 (MRC Dundee), rabbit anti-UBXN6 (ab221167; Abcam), rabbit anti-FAF2 (16251-1-AP; Proteintech), rabbit anti-PLAA (PA5-54299; Invitrogen), rabbit anti-RARA (ab275748; Abcam), and rabbit anti-LC3 A/B (4108S; Cell signaling).

Techniques: Western Blot, Molecular Weight

Journal: International Journal of Endocrinology

Article Title: Ginsenoside Rg1 Improves Inflammation and Autophagy of the Pancreas and Spleen in Streptozotocin-Induced Type 1 Diabetic Mice

doi: 10.1155/2023/3595992

Figure Lengend Snippet: The expression of CXCL16, NF- κ B, TF, LC3, and P62 proteins in the pancreas and spleens. (Mean ± SD, n = 3) (a–f) the expression of CXCL16, NF- κ B, TF, LC3, and P62 proteins in the pancreas. (g–l) The expression of CXCL16, NF- κ B, TF, LC3, and P62 proteins in the spleens. The improvement of Rg1 treatment on type 1 diabetes mice was related to the increase of CXCL16, NF- κ B, and TF and the raised autophagy (IL3 II/LC 3 I). ∗∗ p < 0.01, vs. the control group; ns p > 0.05, ## p < 0.01 vs. the DM group.

Article Snippet: And NF- κ B (1 : 1000, 6956T), LC3 (1 : 1000, 4599T), and P62 (1 : 1000, 5114T) antibodies were provided by Cell Signaling Technology, Inc. (MA, US).

Techniques: Expressing

Journal: bioRxiv

Article Title: Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

doi: 10.1101/2023.03.17.533187

Figure Lengend Snippet: AML12 cells transfected with NCLX (siNCLX), MCU (siMCU) or negative control (siNC) siRNAs were analyzed by ( A-C ) western blot for levels of LC3 I and LC3 II proteins 72 hours after transfection, under complete media (CM) conditions and in the absence (Basal) or presence of 100 nM Bafilomycin A1 (+BafA1) for 1 hour (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Bonferroni’s multiple comparisons test. ( D ) LC3 II net flux calculated as the difference in LC3 II levels between samples treated with Bafilomycin A1 minus untreated samples (n = 6). Data were analyzed by one-way RM ANOVA followed by Bonferroni’s multiple comparison test. ( E ) RT-qPCR for LC3A and LC3B mRNA, 48 and 72 hours after transfection (n = 5-6). Data were analyzed by two-tailed paired t tests. ( F,G ) Representative images and quantification of autophagosomes (mCherry + /GFP + ; yellow puncta) and autolysosomes (mCherry + /GFP - ; red puncta) per cell in AML12 cells stably expressing the mCherry-EGFP-LC3B probe, 72 hours after siRNA transfection (n = 4). Data were analyzed by two-way randomized blocks ANOVA followed by Bonferroni’s multiple comparisons test. ( F ) Western blot analysis for total polyubiquitinated proteins (Ub) in AML12 cells 72 hours after siRNA transfection in complete media (CM) under basal conditions (n = 6) or ( H ) after treatment with 100 nM Bafilomycin A1 (+BafA1, I ) for 1 hour (n = 4). Coomassie staining of the membrane was used as a loading control. Data were analyzed by one-way RM ANOVA followed by Bonferroni’s multiple comparison test. Bar graphs show mean ± SD. Dots in each group represent biological replicates and dots of equal color indicate paired replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant.

Article Snippet: Primary antibodies against LC3 A/B (12741; 1:1,000), LC3B (; 1:1,000), ATG7 (8558; 1:1,000), ATG12 (4180; 1:1,000), ULK1 (8054; 1:500), p-ULK S555 (5869, 1:500), p-ULK S757 (14202; 1:500), 4E-BP1 (9644; 1:1,000), p-4E-BP1 (2855; 1:1,000), MCU (14997; 1:1,000) and α-tubulin (3873; 1:10,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Negative Control, Western Blot, Quantitative RT-PCR, Two Tailed Test, Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

doi: 10.1101/2023.03.17.533187

Figure Lengend Snippet: AML12 cells transfected with NCLX (siNCLX) or negative control (siNC) siRNA were analyzed by ( A ) western blots for LC3 II and ( B ) LC3 I after 1 hour exposure to either complete media (CM) or HBSS (n = 6). ( C ) Western blot analysis of LC3 II after 1 hour exposure to HBSS in the absence or presence of 100 nM Bafilomycin A1 during the last 30 min of treatment (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Tukey’s multiple comparisons test. ( D ) LC3 II net flux in siNC and siNCLX cells treated with HBSS. Data were analyzed by two-tailed unpaired t test. ( E ) western blots for LC3 II and ( F ) LC3 I after 3 hours exposure to complete media (CM) in the presence of vehicle (DMSO) or 200 nM rapamycin (n = 6). ( G ) Western blot analysis of LC3 II after 3 hours of 200 nM rapamycin treatment in the absence or presence of 100 nM Bafilomycin A1 during the last hour of treatment (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Tukey’s multiple comparisons test. ( H ) LC3 II net flux in siNC and siNCLX cells treated with 200 nM rapamycin. Data were analyzed by two-tailed unpaired t test. ( I ) Representative images and quantification of autophagosomes (mCherry + /GFP + ; yellow puncta) and autolysosomes (mCherry + /GFP - ; red puncta) per cell in AML12 stably expressing the mCherry-EGFP-LC3B probe, after 1 hour exposure to HBSS (n = 4). Data were analyzed by two-tailed unpaired t test. Data are presented in columns with means or means ± SD. Dots in each group represent biological replicates and paired biological replicates are represented by equally colored symbols. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant.

Article Snippet: Primary antibodies against LC3 A/B (12741; 1:1,000), LC3B (; 1:1,000), ATG7 (8558; 1:1,000), ATG12 (4180; 1:1,000), ULK1 (8054; 1:500), p-ULK S555 (5869, 1:500), p-ULK S757 (14202; 1:500), 4E-BP1 (9644; 1:1,000), p-4E-BP1 (2855; 1:1,000), MCU (14997; 1:1,000) and α-tubulin (3873; 1:10,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Negative Control, Western Blot, Two Tailed Test, Stable Transfection, Expressing

Journal: PeerJ

Article Title: Roles of cuproptosis-related gene DLAT in various cancers: a bioinformatic analysis and preliminary verification on pro-survival autophagy

doi: 10.7717/peerj.15019

Figure Lengend Snippet: (A) GSEA analysis on DLAT-related genes with autophagy. (B) After Huh7 and HepG2 cells were transfected with siNC or siDLAT, the protein level of DLAT protein was detected by western blot. (C) Huh7 and HepG2 cells transfected with siNC or siDLAT were analyzed in colony formation assays, and the p values were as follows: 0.0068, 0.0047, 0.000, 0.000. (D) Huh7 and HepG2 cells were transfected with siNC or siDLAT, following up with detection of DLAT, LC3 I/II and Beclin-1. (E) After Huh7 and HepG2 cells were transfected with siNC or siDLAT, the formation of autophagosome was observed under by TEM method, and the p values were as follows: 0.0418, 0.0404, 0.0452, 0.0131. * P < 0.05; ** P < 0.01.

Article Snippet: PVDF membranes were sealed with 5% skimmed dry milk for 1 h and then incubated with 1:1000 primary antibodies of DLAT (Signal Antibody, Greenbelt, MD, USA), LC3 I/II (CST, Danvers, MA, USA), Beclin-1 (CST, Danvers, MA, USA) and GAPDH (Proteintech, Wuhan, China) at 4 °C overnight.

Techniques: Transfection, Western Blot