Fig 2: There is a crosstalk between ER stress and necroptosis in PA-induced cardiomyocyte hypertrophy. (a) Treating the NCMs with PA (200 µM) and OA (250 µM), the mRNA expressions of GRP78, ATF6, and CHOP were increased in PA group, not OA group, n = 4. (b) Pretreating the H9c2 cells with pravastatin (10 µM) and effectively blocking the upregulated mRNA expression of GRP78, ATF6, and CHOP (ER stress markers) induced by PA and thapsigargin (100 nM) (an ER stress agonists) via real-time PCR, n = 3. (c) Pretreating the H9c2 cells with pravastatin (10 µM) and also effectively blocking the upregulated mRNA expression of RIPK1/RIPK3 induced by PA and thapsigargin via real-time PCR, n = 3. (d) The increased protein level of GRP78 and CRT in NCMs was downregulated by pravastatin (10 µM) (western blot), n = 3. Data in (a), (b), (c), and (d) are expressed as mean ± SD, * indicates p < 0.05 compared to control treatment, and # indicates p < 0.05 compared to PA treatment.

Fig 3: Chaperone proteins calnexin (CANX) and calreticulin (CALR) regulate SARS-CoV-2 viral entry.(A) Parent Spike was transfected into WT, CANX-KO, and CALR-KO 293Ts. Spike expression was similar in all cells based on anti-FLAG that detects the Spike C terminus (left). A partial (10 to 25%) reduction in anti-RBD Ab/Spike S1 association (middle) was noted, and this correlated with ACE2-Fc binding (right). (B) 293Ts were transfected with parent Spike. Spike immunoprecipitated from cell lysate 24 hours after transfection using anti-FLAG mAb and coprecipitated both CANX and CALR. CALR (top), CANX (middle), and Spike (bottom) were analyzed both in the immunoprecipitated (protein A/G “bead”) sample and the supernatant remaining after bead removal. Arrows and boxes indicate target proteins. (C) All Spike mutants coprecipitated CANX in the anti-FLAG Spike pulldown assay. Cells without Spike (“untransfected”) served as negative control. (D) Parent pseudotyped lentivirus was produced in WT, CANX-KO, and CALR-KO. Western blot showed reduced Spike incorporation into virions produced in both KOs. (E) Pseudovirus entry into 293T/ACE2, measured 72 hours after infection, was reduced when virus were produced in CANX or CALR-KO 293Ts. (F) VSV-G pseudotyped lentivirus entry into 293T/ACE2 was not reduced by either CANX or CALR. (G) Luc-VLP containing parent Spike were produced in WT, CANX-KO, and CALR-KO 293Ts. Luc-VLPs produced in CANX-KO displayed reduced entry into A549-ACE2-TMPRSS2, Calu-3, and 293T/ACE2 cells. Luc-VLP viral entry is reported on the basis of luminescence signal, normalized to parent levels in each run. No spike Luc-VLP lacks Spike. Data are means ± SD (n = 3). *P < 0.05 with respect to all other treatments.

Fig 4: Overexpression of constitutively active ATF6a in cultured mouse detrusor cells increases Pdi, Calr, Sdf2l1 and Manf.Cells were transduced with Ad-CMV-null or Ad-CMV-ATF6a (amino acids 1–373) and ATF6a target proteins as well as smooth muscle marker proteins were examined by western blotting (a). Panels b through (e) show summarized data for Pdi, Calr, Sdf2l1 and Manf. No effect was seen on any of the contractile markers (summarized data in Supplemental Fig. 3a–d) or on GM130. Panel f shows qRT-PCR for myocardin and two of the contractile markers (calponin: Cnn1, tropomyosin: Tpm1). Panels g and h show bladder smooth muscle cells cultured on membrane inserts and transduced with Ad-CMV-null and Ad-CMV-ATF6a, respectively. ER cisterns are labeled blue and bars represent 1 µm.

Fig 5: 4D7 disrupts interaction with TpoR and downstream signalling Cell extracts blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin from TF-1 TpoR cells after incubation with 10 or 20 µg/ml 4D7 or IgG for 4 or 8 h, in presence of TPO. The last line indicates TPO withdrawal.Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells.Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 h.Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 h. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.Fraction of apoptotic sub-G0 population of TF-1 TpoR CALRdel61 cells after 48 h of 4D7 or IgG treatment (n = 3 biological replicates, bars represent standard deviation for three replicates, normalized to IgG, with a Student's unpaired t-test used to determine statistical significance).Western blot showing caspase 3 cleavage occurring within 24 h of TPO withdrawal in TPO-dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 h of treatment with 20 µg/ml 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-h treatment with 20 µg/ml 4D7 but not PBS or 20 µg/ml IgG. CALR monomers and dimers are detectable by polyclonal anti-wild-type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wild-type CALR protein; asterisk, non-specific bands. Source data are available online for this figure.