Fig 2: Reduced expression of CHIP sensitizes ErbB2-overexpressing breast cancer cell lines to ER stress. (A) SKBR3 and 21MT1 cells were seeded in six-well plates and incubated with ER stress inducer thapsigargin (50nM) for 24 h. Cleared lysates (50 µg protein/lane) were left untreated or treated with lambda protein phosphatase (400U) for 30 min and then subjected to Western blotting for GRP78, PERK and Hsc70 (loading control). (B,C) SKBR3 (B) and 21MT1 (C) cells were seeded in six-well plates and incubated with indicated concentrations of thapsigargin for 24 h. Cleared lysates (50 µg/lane) were subjected to Western blotting for ErbB2, GRP78, phospho-eIF2a, PERK, CHIP and Hsc70 (loading control). (D) SKBR3 and 21MT1 cells were seeded in soft agar (0.6% bottom layer, 0.35% top layer) in six-well plates and treated with the indicated concentrations of thapsigargin for 2 weeks. Data represent mean ± SD, n = 3, * p < 0.05.

Fig 3: NRG1/ERBB3/ERBB2-dependent growth of differentiating spermatogonia. (a) ATRA and Neuregulin-1 stimulate development of aligned spermatogonia. tgGCS-EGFP+ spermatogonial scored/well after 1 week of culture in SGF Medium (Table 1) on laminin and then 6 additional days with and without ATRA (3 µM) in combination with GDNF (0.1 nM) and/or NRG1 (5 nM). Representative of duplicate in vitro spermatogenesis colony-forming assays scored after 2 days in SGF Medium and 6 days in SD Medium (±S.E.M., triplicate wells). (b) Effects of various ERBB-family receptor agonists on soma-free rat spermatogonial cultures on laminin. Respective ERBB agonists were substituted for NRG1 in SD Medium. Representative of duplicate in vitro spermatogenesis colony forming assays (±S.E.M., triplicate wells). Inset: western blotting for pAKT-S473 (arrowhead) and p42/44 (lower bands) in spermatogonia in SGF Medium for 2 days, prior to culturing with and without ATRA for 4 days, and then treating with and without NRG1 for 20 min before lysing. (c) ERBB2 inhibitors disrupt NRG1-dependent spermatogonial development on laminin. Representative of duplicate colony-forming assays scored (±S.E.M., triplicate wells). (d) Images of NRG1-dependent syncytial growth of tgGCS-EGFP+ spermatogenic cells on laminin. Scale, 100 µm. Nuclei labeled with Hoechst 33342 dye.

Fig 4: Bortezomib induces ER stress in ErbB2-overexpressing breast cancer cell lines and exhibits synergistic inhibition of cell proliferation when combined with Trastuzumab. (A,B) 21MT1 (A) and SKBR3 (B) cells were seeded in six-well plates and incubated with Bortezomib for 24 h. Cleared lysates (50 µg/lane) were subjected to Western blotting for ErbB2, GRP78, phospho-eIF2a, PERK, CHIP and Hsc70 (loading control). (C–E) BT474 and SKBR3 cells were seeded in 96-well plates and cultured for 5 days in the presence of either Trastuzumab or Bortezomib alone or combinations of the two at various concentrations. Viable cells were stained with the MTT dye and optical density read at 570 nm wavelength in a plate reader. Data represent mean ± SD, n = 3. (F) Combination index was calculated using the CalcuSyn software (http://www.biosoft.com/w/calcusyn.htm; accessed on 16 July 2021 per the method of T.C. Chou and Mike Hayball).

Fig 5: CHIP regulates cell surface ErbB2 levels. (A) Western blot analysis of SKBR3 and 21MT1 cell lysate for CHIP, ErbB2, phospho-Akt and total Akt levels; ß-actin was used as a loading control. (B) SKBR3 and 21MT1 cell surface ErbB2 level was determined by FACS analysis. Representative FACS profiles are shown in top panel. Quantification of surface ErbB2 levels (based on relative change in mean fluorescence intensity with control shRNA as 1) is shown in the bottom panel. Data represent mean ± SEM, n = 3. * p < 0.05. (C) SKBR3 control shRNA, CHIP shRNA, MSCV-puro and MSCV-CHIP cells were seeded on glass coverslips, fixed, and stained with anti-ErbB2 antibody followed by staining with the corresponding fluorescent secondary conjugates. Coverslips were mounted with anti-fade containing DAPI and images were acquired under a Zeiss LSM 710 confocal microscope.