Journal: Journal of Cellular and Molecular Medicine

Article Title: Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain‐mediated cleavage of Atg5/ LAMP2

doi: 10.1111/jcmm.17642

Figure Lengend Snippet: Calpain activation is involved in autophagic flux impairment and diabetic MIRI. (A,B) Cardiac calpain‐1 and calpain‐2 expression during SZT‐induced diabetes, I/R and diabetic I/R by Western blotting and quantitative analyses ( n = 6 per group). (C) Calpain activity by N‐succinyl‐LLVY‐AMC fluorometry of the four wild‐type mice groups and corresponding Tg‐CAST mice groups. (D) Relative LC3II expression by Western blot and quantitative analyses in wild‐type mice and Tg‐CAST mice under STZ and I/R stress with or without CQ treatment. (E) P62 expression by Western blot and quantitative analyses. (F,G) Representative images of Evans blue‐TTC staining of heart samples of WT mice and Tg‐CAST mice with diabetic I/R and quantitative analyses of ratio of myocardial infarction to area at risk ( n = 6 per group). (H,I) Representative images of echocardiography of WT mice and Tg‐CAST mice with diabetic I/R and analyses of LVEF. (J) Calpain activity of cultured cardiomyocytes under H/R and(or) H/R stress with or without PD150506 treatment ( n = 6 per group). (K) Viability of cultured cardiomyocytes by CCK8. (L,M) Representative images of laser confocal microscopy of mCherry‐GFP‐LC3 puncta in cultured mice cardiomyocytes of the eight groups and quantitative analyses of autophagosomes (yellow dots) and autolysosomes (red dots) per cell. (Scale bar = 10 μm, n = 30 cells per group). * p < 0.05; ** p < 0.01; *** p < 0.001; # p > 0.05.

Article Snippet: Primary antibodies include the following: LC3 (1:1000, 4108, CST), P62 (1:1000, 23,214, CST), calpain‐1 (1:1000, 2556, CST), calpain‐2 (1:1000, 2539, CST), Beclin‐1 (1:1000, 3495, CST), Atg5 (1:1000, 2630, CST), LAMP2 (1:1000, ab13524, Abcam), β‐ACTIN (1:1000, 3700, CST).

Techniques: Activation Assay, Expressing, Western Blot, Activity Assay, Staining, Cell Culture, Confocal Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain‐mediated cleavage of Atg5/ LAMP2

doi: 10.1111/jcmm.17642

Figure Lengend Snippet: Calpain‐mediated downregulation of Atg5 and LAMP2 during diabetic MIRI. (A,B) Expression levels (corrected by β‐Actin level) of LAMP2 and Atg5 and their cleaved fragments during SZT‐induced diabetes, I/R and diabetic I/R by Western blotting and quantitative analyses. (C,D) Expression levels (corrected by β‐Actin level) of calpain‐1, calpain‐2, LAMP2, Atg5 and their cleaved fragments of wild‐type and Tg‐CAST mice underwent STZ plus I/R stress by Western blotting and quantitative analyses. (E) mRNAs levels of Atg5 and LAMP2 by RT‐qPCR of wild‐type mice hearts subjected to various treatment. (F,G) Representative images of LAMP2 (red) and Atg5 (green) expression of cultured cardiomyocytes subjected to HG and H/R with and without calpain inhibitor PD150606 by immunofluorescent staining (nuclei were located by DAPI (blue), Fluorescence 525/600 nm, arbitrary units, scale bar = 20 μm). and quantitative analysis (n = 30 cells per group). * p < 0.05; ** p < 0.01; *** p < 0.001; # p > 0.05. ( n = 6 per group).

Article Snippet: Primary antibodies include the following: LC3 (1:1000, 4108, CST), P62 (1:1000, 23,214, CST), calpain‐1 (1:1000, 2556, CST), calpain‐2 (1:1000, 2539, CST), Beclin‐1 (1:1000, 3495, CST), Atg5 (1:1000, 2630, CST), LAMP2 (1:1000, ab13524, Abcam), β‐ACTIN (1:1000, 3700, CST).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Combination Therapy with Gossypol Reveals Synergism against Gemcitabine Resistance in Cancer Cells with High BCL-2 Expression

doi: 10.1371/journal.pone.0050786

Figure Lengend Snippet: Immunoblot of anti-apoptotic (pAKT and PI3KR2) and pro-apoptotic (BAD, CASP6 and CAPN1) genes in GEM-R cell line and GEM-S cell lines. Cells were treated with gemcitabine and combination with gossypol for 48 hours. The results shown are representative of two independent experiments.

Article Snippet: The respective primary antibodies were used as follows: anti-Bcl-xl (Cell Signaling; MA, US) at 1∶1000 dilution, anti-Bad (Cell Signaling; MA, US) at 1;1000 dilution, anti-Bak (Cell Signaling; MA, US) 1∶1000 dilution, anti-Bax (Cell Signaling; MA, US) 1∶3000 dilution, anti-caspase-6 (Cell Signaling; MA, US) 1∶1000 dilution, anti-P1K3R2 (Cell Signaling; MA, US) 1∶1000 dilution, anti-pAKT (Cell Signaling; MA, US) at 1∶1000 dilution, anti-calpain-1 (Cell Signaling; MA, US) at 1∶1000 dilution, anti-Actin (loading control; Cell Signaling; MA, US) at 1∶3000 dilution, anti-Bcl-2 (Santa Cruz Biotechnology; CA, USA) 1∶3000 dilution, and anti-Mcl-1 (Santa Cruz Biotechnology; CA, USA) at 1∶3000 dilution, and anti-Noxa (Calbiochem, Merck; Germany) 1∶1000 dilution.

Techniques: Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Aloe-Emodin Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Colorectal Cancer Cells

doi: 10.12659/MSM.908400

Figure Lengend Snippet: Aloe-emodin modulated ER stress involving in cytosolic calcium overload and calpain/caspase-12 pathway in SW620 and HT29 cells. ( A ) SW620 cells and ( B ) HT29 cells were treated with indicated concentrations of Aloe-emodin for 24 hours, then stained with Fluo4-AM (2 μM); the cytosolic calcium levels were quantitatively analyzed through flow cytometry; ( C–F ) The calpain-1, calpain-2, and caspase-12 proteins expression in SW620 cells were analyzed by western blot; ( G–J ) The calpain-1, calpain-2, and caspase-12 proteins expression in HT29 cells were analyzed by western blot. * P <0.05, ** P <0.01, *** P <0.001, as compared with the control group; n=4.

Article Snippet: The primary antibodies included: Bcl-2 (1: 2000, Epitmics, USA), Bax (1: 1000, CST, USA), GRP78 (1: 1000, CST, USA), p-PERK (1: 1000, CST, USA), p-eIF2α (1: 1000, CST, USA), CHOP (1: 1000, CST, USA), calpain-1 (1: 1000, CST, USA), calpain-2 (1: 1000, CST, USA) and caspase-12 (1: 1000, CST, USA).

Techniques: Staining, Flow Cytometry, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress

doi: 10.3389/fcell.2022.1008542

Figure Lengend Snippet: Vimentin cleavage is blocked by inhibitors of the proteasome and calpains, which are elevated in GAN. (A) Immunoblotting for vimentin (top) and actin (bottom; loading control) in Triton X insoluble pellet fractions from untreated (lanes 1–3, 7–9) or water-exposed (lanes 4–6, 10–12) control 56.3 and GAN 56.1 fibroblasts in the absence or presence of the DMSO vehicle control, calpain inhibitor (MDL-28170), or calpain/proteasome inhibitor (MG-132). Note partial cleavage inhibition by MDL and near-complete inhibition by MG-132. (B) Immunoblotting for vimentin (top) and actin (bottom; loading control) in reduced Triton X insoluble fractions and for calpain-1 (middle top), calpain-2 (middle), and calpastatin (middle bottom) in reduced Triton X soluble fractions from GAN and control fibroblasts exposed to 8 min of hypotonic stress and treated with the DMSO vehicle control or MG-132. Shown are technical replicates from an individual experiment. The experiment was independently repeated at least 3 times. (C) Quantification of the relative fragment 1 (FR1) and fragment 2 (FR2) band intensities normalized to the full length vimentin band for each cell line (GAN 56.1 fibroblasts; control 56.3 fibroblasts) and treatment group (from panel B). **** p < 0.0001; two-way ANOVA. (D) Quantification of calpain-1, calpain-2, and calpastatin levels (from panel B). ** p < 0.01; **** p < 0.0001; two-way ANOVA.

Article Snippet: The following primary antibodies and concentrations were utilized: rabbit anti-Vimentin (Cell Signaling Technology, D21H3, WB 1:1,000, IF 1:200), mouse anti-Vimentin (Invitrogen, V9, WB 1:1,000–1:2,500, IF 1:100), mouse anti-Actin (Thermo Fisher Scientific, ACTN05, WB 1:1,000), mouse anti-Actin (Santa Cruz, SPM161, WB 1:1,000), rabbit anti-Calpastatin (Cell Signaling Technology, WB 1:1,000), rabbit anti-Calpain 1 (Cell Signaling Technology, large subunit Mu-type, WB 1:1,000), rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, WB 1:1,000), and rabbit anti-Calpain 2 (Cell Signaling Technology, large subunit M-type, E3M6E, IF 1:200).

Techniques: Western Blot, Inhibition