Journal: Cancer Science

Article Title: R eticulocalbin 3 : A Ca 2+ homeostasis regulator that promotes esophageal squamous cell carcinoma progression and cisplatin resistance

doi: 10.1111/cas.15487

Figure Lengend Snippet: Reticulocalbin3 (RCN3) regulated MMP‐2 and MMP‐9 expression through inositol 1,4,5‐trisphosphate receptor 1 (IP3R1)–Ca 2+ –calcium/calmodulin‐dependent protein kinase (CaMKII). (A) In esophageal squamous cell carcinoma (ESCC) cell lines, RCN3 is positively correlated with IP3R1 expression. * p < 0.05, Pearson's correlation coefficient. (B) Co‐IP analysis showed that RCN3 interacted with IP3R1. (C) Immunofluorescence staining showed there the expression and localization of RCN3 and IP3R1 in ECA109 cells. (D–G) Fluo‐4 AM showed intracellular Ca 2+ concentration. (H) Western blot analysis the expression of related proteins of the IP3R1–Ca 2+ –CaMKII–c‐Jun signaling pathway. (I) Expression of MMP‐2 and MMP‐9 inhibited by IP3R–Ca 2+ –CaMKII blockers in OV‐RCN3 ESCC cells. (J) Invasion ability of OV‐RCN3 ESCC cell inhibited by IP3R–Ca 2+ –CaMKII blockers. * p < 0.05, ** p < 0.01, *** p < 0.001, t ‐test. NC, negative control

Article Snippet: The membranes were blocked in 5% skim milk for 2 h at room temperature then incubated with primary Abs overnight at 4°C: GAPDH (Proteintech, 10494‐1‐AP), β‐tubulin (Proteintech, 10094‐1‐AP), RCN3 (Abcam, ab204178), HIF‐1α (Proteintech, 20960‐1‐AP), IP3R1 (Santa Cruz Biotechnology, sc‐271197), CaMKII (GeneTex, GTX52377), p‐CaMKII (Santa Cruz Biotechnology, sc‐32289), c‐Jun (Santa Cruz Biotechnology, sc‐166540), p‐c‐Jun (Santa Cruz Biotechnology), MMP2 (Proteintech, 66366‐1‐Ig), and MMP9 (Proteintech, 10375‐2‐AP).

Techniques: Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Concentration Assay, Western Blot, Negative Control

Journal: Cancer Science

Article Title: R eticulocalbin 3 : A Ca 2+ homeostasis regulator that promotes esophageal squamous cell carcinoma progression and cisplatin resistance

doi: 10.1111/cas.15487

Figure Lengend Snippet: Reticulocalbin3 (RCN3) promoted chemoresistance by targeting inositol 1,4,5‐trisphosphate receptor 1 (IP3R1)/Ca 2+ through inhibiting reactive oxygen species (ROS) production. (A) Western blot showed the expression of chemoresistance proteins MDR1(D‐11), ABCG2, and RCN3. (B) After treatment with different concentrations of cisplatin (DDP), CCK‐8 showed the cell growth inhibition rate. (C) CCK‐8 showed the survival rate of drug‐resistant cells after interfering RCN3 expression. (D) Transmission electron microscopy showed the structure of ECA109, ECA109/DDP, and ECA109/DDP‐shRCN3 cells. (E) Apoptosis analysis showed apoptosis rates of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (F) Quantitative analysis of mitochondrial membrane potential measurements with JC‐1. The change of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (G) ROS analysis showed reactive oxygen of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (H) Apoptosis analysis showed apoptosis rate of overexpression of RCN3 ± siIP3R1, treated with DDP for 24 h. (I) Quantitative analysis of mitochondrial membrane potential measurements with JC‐1. The change of overexpression of RCN3 ± siIP3R1 resistant cell line, treated with DDP 24 h. (J) ROS analysis showed overexpression of RCN3 ± si‐IP3R1, treated with DDP 24 h. (K) Immunohistochemical staining of RCN3 protein in esophageal squamous cell carcinoma (ESCC) biopsy tissues (left panel). H&E staining of platinum‐based chemotherapy ESCC tissues (right panel). * p < 0.05, ** p < 0.01, *** p < 0.001, t ‐test. NC, negative control

Article Snippet: The membranes were blocked in 5% skim milk for 2 h at room temperature then incubated with primary Abs overnight at 4°C: GAPDH (Proteintech, 10494‐1‐AP), β‐tubulin (Proteintech, 10094‐1‐AP), RCN3 (Abcam, ab204178), HIF‐1α (Proteintech, 20960‐1‐AP), IP3R1 (Santa Cruz Biotechnology, sc‐271197), CaMKII (GeneTex, GTX52377), p‐CaMKII (Santa Cruz Biotechnology, sc‐32289), c‐Jun (Santa Cruz Biotechnology, sc‐166540), p‐c‐Jun (Santa Cruz Biotechnology), MMP2 (Proteintech, 66366‐1‐Ig), and MMP9 (Proteintech, 10375‐2‐AP).

Techniques: Western Blot, Expressing, CCK-8 Assay, Inhibition, Transmission Assay, Electron Microscopy, Membrane, Over Expression, Immunohistochemical staining, Staining, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Loss of parkin increases endoplasmic reticulum (ER)-mitochondria contact sites. (A) Confocal microscopy was used for live cell imaging of both control cells (upper) and Parkin KO M17 cells (bottom). ER was stained with an ER-Tracker (green) and mitochondria were stained with MitoTracker (red) (Scale bar, 20 μm). Quantification of ER-mitochondria association was performed using ImageJ (bar graphs on the right). (B) Confocal microscopy images of proximity ligation assays (PLA) of ER-mitochondria association. PLA signals (red dots) were determined by interaction between IP3R and VDAC1 in control cells (upper) and Parkin KO M17 cells (bottom) (Scale bar, 20 μm). PLA red fluorescent dots were present as the number of positive interactions per nucleus (bar graphs on the right). (C) Electron micrographs of mitochondria connected to the ER (pseudocolored green) in control and Parkin KO M17 cells (Scale bar, 1 μm). (D-F) Ultrastructural analysis of ER-mitochondria association. Quantitative analysis of the percentage of the mitochondria-associated endoplasmic reticulum membrane (MAM) to mitochondria (D), type of MAMs apposition (F) and average length (I) of ER-mitochondria association in control cells (n = 7) and Parkin KO M17 cells (n = 8). (G) Electron micrographs of mitochondria connected to the ER (pseudocolored green) in neurons from wild-type (WT) and Parkin KO mouse brains. (H-J) Ultrastructural analysis of ER-mitochondria association. Quantitative analysis of the percentage of the MAM to mitochondria (H), type of MAM apposition (I), and average length (J) of ER-mitochondria association (n = 5 mice per group, with 4 neurons per mouse). Data are expressed as means ± SEM based on three independent experiments. Data were analyzed using a two-tailed unpaired Student's t-test. *P < 0.05; **P<0.01; ***P < 0.001.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Confocal Microscopy, Live Cell Imaging, Control, Staining, Ligation, Membrane, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Parkin resides in the MAM and interacts with the IP3R-Grp75-VDAC1 complex. (A-B) Subcellular fractionation and immunoblot characterization of parkin in the MAM fraction in wild-type C57BL6 mouse brain (A) and parkin-overexpressing M17 cells (B). Cells were fractionated into whole lysates (WL), crude mitochondria (CM), pure mitochondria (PM), ER, and MAM. Three independent experiments were conducted. (C) In situ close association between Parkin/IP3R (left), Parkin/Grp75 (middle), and Parkin/VDAC1 (right) were determined by proximity ligation assay (PLA) in normal M17 cells (Scale bar, 20 μm). (D-F) Immunoblot analysis of IP3R, Grp75, and VDAC1 in the parkin immunoprecipitates of total lysates from parkin-overexpressing M17 cells (D) and ventral midbrain of mouse brain (E). MAM fractions were prepared from the ventral midbrain of the mouse brain (F). Three independent experiments were conducted. (G-H) Blue native-polyacrylamide gel electrophoresis (BN-PAGE) and immunoblot analysis of CM fractions prepared from parkin-overexpressing M17 cells (G) and ventral midbrain of mouse brain (H) showed a large protein complex. The red box in G-H highlights the macrocomplex that contains all four components. Three independent experiments were conducted. (I) Two-dimensional separation and immunoblot analysis of CM fraction from parkin-overexpressing M17 cells showed a large protein complex. The red box in I highlights the macrocomplex that contains all four components. Three independent experiments were conducted.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Fractionation, Western Blot, In Situ, Proximity Ligation Assay, Polyacrylamide Gel Electrophoresis

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Calcium-Dependent Localization of Parkin at the MAM. (A) Representative images of PLA targeting IP3R-Parkin interactions after treatment with 2-APB (50 μm, 2 hours) in M17 cells (Scale bar, 20 μm). (B) Representative immunoblot and quantitative analyses of IP3R coimmunoprecipitated with parkin after treatment with 2-APB (50 μm, 2 hours) in overexpressing WT parkin M17 cells. Three independent experiments were conducted and were quantified. (C) BN-PAGE of the complex after treatment with 2-APB (50 µm, 2 hours). Representative immunoblot images were detected by parkin. Three independent experiments were conducted. (D) Immunoblot analysis of MAM proteins in the WL and MAM fractions from control and 2-APB treatment (50 µm, 2 hours). Proteins were normalized to calreticulin. Three independent experiments were conducted and were quantified. Data are expressed as means ± SEM based on three independent experiments. Data were analyzed using a two-tailed unpaired Student's t-test. *P < 0.05; **P<0.01; ***P < 0.001.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Parkin regulates the stability of the IP3R-Grp75-VDAC1 complex. (A) Immunoblot analysis of chosen proteins in control and Parkin KO M17 cells. Control and Parkin KO cells were fractionated into WL and CM. Cytochrome c oxidase subunit IV (COX IV) was used as a loading control for CM proteins. Three independent experiments were conducted and were quantified. (B) Immunoblot analysis of MAM proteins in the WL and MAM fractions from WT and Parkin KO mice (n = 6/group). Proteins were normalized to calreticulin. (C) Immunoblot analyses of time-dependent degradation of IP3R were detected after treatment with cycloheximide (CHX, 100 μg/ml, 0-4 hours) in control and Parkin KO M17 cells. Three independent experiments were conducted and were quantified. (D) BN-PAGE analysis of the macrocomplex detected by IP3R in crude mitochondria from control and Parkin KO M17 cells. SDS-PAGE immunoblot was used to analyze parkin in CM. Data are quantified from three independent experiments. (E) BN-PAGE and immunoblot analysis of the macrocomplex detected by IP3R in the crude mitochondria fraction from mouse brain homogenates (left). Representative immunoblot images detected by parkin (n = 5/group, right). (F) Immunoblot analysis of Grp75/VDAC1 was coimmunoprecipitated with IP3R antibody in control and Parkin KO M17 cells. Three independent experiments were conducted and were quantified. (G) Representative images and quantification of IP3R-VDAC1 PLA signals revealed an association in tyrosine hydroxylase positive (TH+) neurons in the substantia nigra of Parkin KO mice. IP3R-VDAC1 PLA (red) was performed in brain sections and co-stained with TH (green) (n = 5/group), (Scale bar, 20 µm). (H) Immunoblot analysis of Grp75 and VDAC1 was coimmunoprecipitated with IP3R antibody in the brain homogenates of wild-type control and Parkin KO mice (n = 5/group). Data are expressed as means ± SEM. Data were analyzed using two-tailed unpaired Student's t-test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, SDS Page, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Parkin tunes the degradation of IP3R through K48-linked ubiquitination. (A) Immunoblot analyses of IP3R protein showed its reduced stability by Parkin-dependent expression in M17 cells. Three independent experiments were conducted and were quantified. (B) Immunoblot analysis was performed to assess IP3R protein expression in M17 cells overexpressing wild-type (WT) parkin, treated with MG132 (20 µM) or 3-MA (2.5 mM) for 24 hours. Three independent experiments were conducted and were quantified. (C) Representative immunoblot and quantitative analyses of the degradation of IP3R from overexpressing vector, WT parkin, and parkin C431S mutant M17 cells. Three independent experiments were conducted and were quantified. (D) Ubiquitination levels of IP3R were assessed in M17 cells expressing WT or C431S parkin plasmid. Cells were treated with 20 µM of MG132 for 4 hours. Three independent experiments were conducted. (E) Ubiquitination levels of IP3R were assessed in M17 cells expressing wild-type (WT) parkin, HA-Ub, and its mutant plasmids. Cells were treated with 20 µM of MG132 for 4 hours. Three independent experiments were conducted. Data are expressed as means ± SEM based on three independent experiments. Data were analyzed using ANOVA with Tukey's multiple comparisons test (A-C). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Plasmid Preparation, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: IP3R is ubiquitinated by Parkin upon induction of E3 activities. (A) Immunoblot analyses of time-dependent degradation of IP3R were detected after treatment with carbonyl cyanide m-chlorophenyl hydrazine (CCCP, 20 µm, 0-6 hours) in control and Parkin KO M17 cells. Three independent experiments were conducted and were quantified. (B) Representative immunoblot and quantitative analyses of IP3R degradation after treatment with CCCP (20 µm, 2 hours) and proteasome inhibitor MG-132 (20 µm, 0.5 hour before CCCP treatment) in M17 cells. Three independent experiments were conducted and were quantified. (C) Representative images of PLA targeting IP3R-Parkin interactions after treatment with CCCP (20 µm, 1 hour) in M17 cells (n = 166-199 cells). (D) Representative immunoblot and quantitative analyses of IP3R coimmunoprecipitated with parkin after treatment with CCCP (20 µm, 1 hour) in overexpressing WT parkin M17 cells. Three independent experiments were conducted and were quantified. (E) BN-PAGE of the complex after treatment with CCCP (20 µm, 2 hours). Representative immunoblot images were detected by IP3R (left) and parkin (right). Three independent experiments were conducted. (F) Ubiquitination levels of IP3R after treatment with CCCP (20 µm, 2 hours) and MG-132 (20 µm, 1 hour before CCCP treatment) in M17 cells. Three independent experiments were conducted. Data are expressed as means ± SEM from three independent experiments. Data were analyzed using a two-tailed unpaired Student's t-test (A, C-F) and ANOVA with Tukey's multiple comparisons test (B). *P < 0.05; **P < 0.01; ***P < 0.0001.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Ubiquitin Proteomics, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Parkin Deficiency Impairs ER-Mitochondria Associations and calcium homeostasis via IP3R-Grp75-VDAC1 Complex

doi: 10.7150/ijbs.121759

Figure Lengend Snippet: Schematic diagram illustrating Parkin-mediated regulation of ER-mitochondria contacts and calcium homeostasis. Under normal conditions, IP3R-mediated Ca²⁺ flux recruits Parkin. Through its E3 ubiquitin ligase activity, Parkin ubiquitinates and degrades IP3R channels. This process leads to disassembly of the IP3R complex and helps regulate mitochondrial calcium homeostasis in mitochondria-associated membranes (MAMs). In the absence of functional Parkin, IP3R complexes abnormally accumulate at MAM regions. This increases cellular sensitivity to calcium stimuli and leads to mitochondrial calcium overload, triggering abnormal opening of the mitochondrial permeability transition pore (mPTP) and ultimately resulting in apoptosis.

Article Snippet: Parkin (sc-32282, 1μg for IP, 1:300 for BN-PAGE), IP3R (sc-377518, 1:100 for PLA, 1μg for IP) and Grp75 (sc-133137, 1:1000 for WB and BN-PAGE, 1:100 for PLA) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Activity Assay, Functional Assay, Permeability

Journal: Cancers

Article Title: Potential Role of PDGFRβ-Associated THBS4 in Colorectal Cancer Development

doi: 10.3390/cancers12092533

Figure Lengend Snippet: Effect of PDGF-D stimulation of THBS4 after blockage of PDGFRβ, IP3R, and STIM1. ( A ) Western blot with anti-THBS4 antibody in whole cell lysate (left panel) or cultured medium (right panel) of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (0, 0.2, 0.5, 1, 2, and 5 μM) for 16 h, 2-APB (0, 5, 10, 20, 50, and 100 μM) for 16 h, or ML-9 (0, 5, 10, 20, 50, and 100 μM) for 16 h, respectively. ( B ) Western blot with anti-THBS4 antibody in whole cell lysate or cultured medium of DLD-1 cells transfected with siIP3R or siSTIM1 and cultured with PDGF-D (20 μM) for 8 h. ( C ) Relative mRNA expression levels as determined with real-time PCR for THBS4 of DLD-1 cells cultured with PDGF-D (20 μM) for 8 h in the presence of imatinib (5 μM) for 16 h, 2-APB (100 μM) for 16 h or ML-9 (100 μM) for 16 h, respectively. Three independent experiments were performed in duplicate. * p < 0.05 when compared with the control t- test.

Article Snippet: DLD-1 cells were transfected with IP3R siRNA 10nM (sc-42475, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA) or STIM1 siRNA 10 nM (sc-76589) along with control siRNA (sc-37007).

Techniques: Western Blot, Cell Culture, Transfection, Expressing, Real-time Polymerase Chain Reaction, Control