Fig 2: ERO1a-mediated stimulation of IP3R activity is critical for ER Ca2+ release. (A) Representative images of p-IP3R showed that OGD treatment increased the phosphorylation of IP3R, which was reversed by EndoA2 overexpression and further strengthened by EndoA2 siRNA knockdown. Scale bars: 50 µm (n=6). (B) Representative images of t-IP3R showed that t-IP3R was not changed under the indicated conditions (n=4). (C) Pretreatment with 2-APB inhibited intracellular Ca2+ release induced by OGD treatment. Scale bars: 50 µm. (n=6). (D-E) Western blotting results showed the expression of cleaved caspase-3, Bax and Bcl-2 after being treated with 2-APB. Densitometric analyses showed that the change of apoptosis-related protein induced by OGD was reversed by pretreatment with 2-APB (n=6, * p <0.05 vs. control, # p <0.05 vs. OGD). (F) qRT-PCR detection of ERO1a mRNA expression. EndoA2 overexpression decreased ERO1a mRNA expression induced by OGD, while EndoA2 siRNA knockdown further increased it (n=6, ** p <0.01 vs. control, ## p <0.01 vs. OGD+Ad-lacZ or OGD+neg). (G) Representative images showed that ERO1a siRNA knockdown attenuated the phosphorylation of IP3R induced by OGD or EndoA2 siRNA knockdown. Scale bars: 50 µm (n=6). (H-I) Western blotting results showed that ERO1a siRNA knockdown reversed the change of apoptosis-related protein induced by OGD or EndoA2 siRNA knockdown (n=6, * p <0.05 vs. control, # p <0.05 vs. OGD+neg, & p <0.05 vs. OGD+EndoA2 si).

Fig 3: GRP75-KD or inhibition accelerated CP-induced mitochondrial dysfunction. (A, C, E, G, I, K) GRP75-expression-changed OC cells were treated with CP (4ug/mL) for 48h. (B, D, F, H, J. L) CP-sensitive and -resistant OC cells were treated with CP (4 µg/mL) and/or MKT077 (40 µM) for 48h. (A, B) iROS: level was determined by Amplite™ ROS Green staining and measured by a microplate reader. (C, D) mtROS level was determined by MitoROS™ 580 staining and measured by a microplate reader. (E, F) The level of intracellular ATP was measured by using the enhanced ATP kit. The ATP level in the NC group of OC cells was set as 100%. (G, H) Intracellular NAD+/NADH ratio was determined by using the NAD+/NADH assay kit. The NAD+ level in NC group of OC cells was set as 100%. (I, J) Change in mitochondrial membrane potential was determined by flow cytometry-based measurement of red fluorescence. The ??m in the NC group of OC cells was set as 100%. (K, L) Quantitative analysis of calcein-AM fluorescent intensity in the presence (quenched) and absence (retained) of CoCl2 in OC cells as determined by flow cytometry. The relative calcein fluorescence in the NC group of OC cells was set as 100%. All data represent mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared to the NC group or untreated OC cells. (M) Working model: GRP75-faciliated MAM integrity represents a checkpoint in regulating the CP-resistance. GRP75-mediated MAM formation boosts ER-mitochondrial Ca2+ fluxes, and drives mitochondrial bioenergetics and ROS production, which control the balance of pro-survival and pro-apoptotic signals in OC cells. (1) Low-level constitutive ER-to-mitochondria Ca2+ fluxes maintain the TCA cycle running, which sustains energy production (ATP), redox homeostasis (NADH generation), anabolic pathways (biosynthesis of macromolecules), and the survival and proliferation of OC cells. (2) CP-exposure elicits the damage to nDNA and mtDNA, causes Ca2+ overload-release from ER to mitochondria through increased MAM formation, leads to pro-apoptotic mtROS production, mPTP opening and cytochrome C release, triggers activation of Caspases, and induces apoptosis in CP-sensitive OC cells. (3) CP-resistant OC cells distinctively manage the [Ca2+]m uptake to stimulate Ca2+-dependent mitochondrial metabolism and pro-survival mtROS level, while avoiding the Ca2+-triggered cell death by fine-tuning GRP75-mediated MAM formation. This probably involves metabolic reprogramming and up-regulated antioxidant enzymes to prevent deleterious [Ca2+]m and mtROS accumulation. (4) GRP75-deficiency abrogates VDAC1-IP3R1 interaction and ER-mitochondrial coupling, causes ER-to-mitochondria Ca2+ transfer-interrupted, mitochondrial dysfunction, compromised OXPHOS (NADH decline), severe bioenergetic crisis (ATP depletion), and eventually results in apoptotic death in CP-sensitive and -resistant OC cells. In these GRP75-depleted cells, CP exposure-induced catastrophic oxidative stress accelerates the mitochondrial dysfunction and cell death.

Fig 4: HSP47 interferes with both calreticulin/SERCA2 interaction and IRE1α/IP3R interaction in pancreatic cancer cells. A, Expression of calreticulin (CALR) and IRE1α protein in human PDAC cell lines. GAPDH was used as an internal control. B, Expression of CALR and IRE1α protein in HSP47 KO PDAC cells (HSP47 KO PANC1 cells and HSP47 KO MIA‐PaCa 2 cells). C, Interaction of HSP47 with CALR and IRE1α in PANC1 cells expressing HSP47‐Myc after treatment with gemcitabine (10 μmol/L). D, Interaction of CALR with SERCA2 in HSP47 KO PANC1 cells expressing SERCA2‐Myc. E, Interaction of CALR with SERCA2 in HSP47 KO PANC1 cells expressing SERCA2‐Myc after treatment with gemcitabine (10 μmol/L). F, Interaction of CALR with SERCA2 in HSP47 KO PANC1 cells expressing SERCA2‐Myc and reconstituting HSP47‐GFP expression after treatment with gemcitabine (10 μmol/L). G, The binding ratio of CALR (IP) /SERCA2‐Myc (IP) was determined by using NIH ImageJ software. *, P < .05. H, Interaction of IRE1α with IP3R in HSP47 KO PANC1 cells expressing IRE1α‐Myc after treatment with gemcitabine (10 μmol/L). (I) Interaction of IRE1α with IP3R in HSP47 KO PANC1 cells expressing IRE1α‐Myc and reconstituting HSP47‐GFP expression after treatment with gemcitabine (10 μmol/L)

Fig 5: Gli1+ cells participate in RME-induced bone formation via IP3R upregulation. (a) Distribution of IP3R+ cells (red), Gli1+ cells (green), and IP3R+ and Gli1+ cells in the control, RME, and RME+GANT61 groups detected with immunofluorescence staining. Scale bar: 100 µm. Regions in boxes are magnified in the right panel: “N” indicates the nasal side of the midpalatal suture and “O” indicates the oral side. Scale bar: 50 µm; n = 3. (b) IP3R+ cells were increased in the RME group and decreased after GANT61 treatment. *P < 0.05; **P < 0.01; n = 3. (c) IP3R+ and Gli1+ cells increased in the RME group and decreased after GANT61 treatment. ***P < 0.005; *P < 0.05; n = 3.