Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Scientific Reports

Article Title: HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior

doi: 10.1038/s41598-021-93807-3

Figure Lengend Snippet: Mass cytometry antibody panel for lung adenocarcinoma.

Article Snippet: HER2 , 148Nd , Surface , 29D8 , Fluidigm , 3148011A.

Techniques: Mass Cytometry

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: ERBB2 (HER2) was the top inhibited upstream regulator in noninvasive fibroblasts. (A) Flow cytometry analysis confirmed the overexpression of SEMA7A in lung fibroblasts. (B) Cell proliferation rates of fibroblasts with SEMA7A overexpression or control fibroblasts were determined by EdU assays. (C) Cell-surface expression of SEMA7A was determined by flow cytometry on single-cell homogenate of CD31 − , CD45 − , EPCAM − cells from IPF and healthy samples. (D) Single-cell Western blot confirmed the downregulation of FOXF1 in invasive fibroblasts. (E and F) Dot plot visualization of the −Log 10 (FDR) (E) and bar plot visualization of the Activation Z-score (F) of the top 30 activated and inhibited upstream regulators of noninvasive fibroblasts by IPA analysis. ERBB2 was the top inhibited regulators of noninvasive fibroblasts. ERBB2 was highlighted as the most inhibited regulator. (G) The regulating network of invasive fibroblasts combining canonical signaling pathways and upstream regulators showed that the core signaling pathway was the invasion of tumor cell lines, suggesting that invasive lung fibroblasts had metastatic cancer-related signatures.

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Flow Cytometry, Over Expression, Control, Expressing, Single Cell Western, Activation Assay, Protein-Protein interactions

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Significantly activated ERBB2 (HER2) signaling pathway in invasive fibroblasts. (A and B) Dot plot visualization of the −Log 10 (FDR) (A) and bar plot visualization of the activation Z-score (B) of the top 30 activated and inhibited upstream regulators of invasive fibroblasts by IPA analysis. ERBB2 was the most activated regulators of invasive fibroblasts. ERBB2 was highlighted as the most activated regulator. (C) IPA analysis revealed the upstream regulators of metastatic lung adenocarcinoma cancer cells compared to primary cancer cells retrieved from GSE131907 . Most of the top activated/inhibited (listed in red/green texts, respectively) upstream regulators of invasive fibroblasts were these of metastatic cancer cells. (D) Pearson correlation analysis of activation z-score of shared upstream regulators ( n = 129) of invasive fibroblasts versus metastatic lung adenocarcinoma cancer cell. Linear regression analysis was performed and visualized in red line. (E) p-HER2, total HER2, and SEMA7A protein levels in sorted SEMA7A high and negative fibroblasts in nine IPF fibroblast lines were determined by Western blot. GAPDH served as loading control. (F) Quantification of the Western blot was used to determine the relative protein levels of p-HER2, total HER2, and SEMA7A in E ( n = 9 per group). (G and H) p-HER2 and total HER2 in sorted fibroblasts from three normal and four IPF lung were determined by Western blot and quantification was performed (H; n = 4). Neg, megative; P, proximal lung regions; D, distal lung regions. Two independent experiments were performed on fibroblasts from different patients (F and H). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student’s t test (F and H). Source data are available for this figure: .

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Activation Assay, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: ERBB2 was the top upstream regulator in fibrotic fibroblasts in human and mouse lungs. (A and B) ERBB2 was the top upstream regulator of both human IPF lung fibroblasts (A) and bleomycin-induced mouse lung fibrotic fibroblasts (B). (C) Sorting strategy for normal and IPF lung fibroblasts for p-HER2 and total HER2 Western blot. (D) Immunostaining of HER2/p-HER2 with activated fibroblast (myofibroblast) marker, α-SMA, in normal and IPF human lung cryosections. IgG isotype was used as control for HER2 and p-HER2 staining. Higher magnifications of the boxed regions were provided. (E) Transcription of ERBB2 (HER2) in myofibroblasts in published scRNA-seq datasets. P value of each comparison, cell number ( n ) of each group, and average expressions of ERBB2 (purple dotted line) were included. (F) Cell proliferation rates of normal fibroblasts with HER2 overexpression or control fibroblasts were determined by EdU assays. (G) Fibroblast viability after Lapatinib treatment was determined by Calcein AM Assay ( n = 8 per group). (H) Fibroblast growth rate after treatment of Lapatinib at increasing concentration ( n = 6 per group). Three independent experiments were performed on fibroblasts from different patients (G and H). Data are the mean ± SEM. ns, not significant by two-way ANOVA (G). Scale bars, 20 μm (D).

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Western Blot, Immunostaining, Marker, Control, Staining, Comparison, Over Expression, Calcein AM Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: HER2 signaling activation increased fibroblast invasion and fibrosis. (A) The expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts were detected by qRT-PCR ( n = 3 per group). (B) Heatmap of the differentially expressed genes of control and HER2 overexpressing normal human lung fibroblasts by bulk RNA-seq. (C) Volcano plot of the top differentially expressed genes between control and HER2 overexpressed normal human lung fibroblasts by bulk RNA-seq. Red dots indicated the genes at Fold_change >0.5 and black dots indicated the genes at Fold_change ≤0.5. (D) Relative expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts detected by bulk RNA-seq ( n = 5 per group). (E) Upregulated cell-surface expression of SEMA7A in HER2 overexpression normal lung fibroblasts was confirmed by flow cytometry analysis. (F and G) Western blotting confirmation of the expression of p-HER2, HER2, SEMA7A, and FOXF1 in HER2 overexpression normal human lung fibroblasts (F) and quantification of the densitometry (G; n = 3 per group). GAPDH served as loading control. (H and I) Representative images (H) and index quantification (I; n = 6 per group) of normal lung fibroblast invasion after HER2 overexpression. (J and K) Trichrome staining (J) and hydroxyproline (K; n = 10 per group) of mice lungs receiving HER2 overexpressing and control normal human lung fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (H) and 500 μm (J). Three or four independent experiments were performed on fibroblasts from different patients (A, D, G, I, and K). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (A, D, G, and I) and two-way ANOVA (K). Source data are available for this figure: .

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Activation Assay, Expressing, Over Expression, Quantitative RT-PCR, Control, RNA Sequencing, Flow Cytometry, Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: HER2 deficiency rescued the dysregulated gene profiles in IPF lung fibroblasts. (A–C) HER2 knockdown efficiency was confirmed by qRT-PCR (A) and Western blotting (B and C; A, n = 8 per group; C, n = 5 per group). (D) Cell-surface protein level of HER2 and SEMA7A in HER2 knockdown IPF lung fibroblasts. (E and F) Representative images (E) and index quantification (F; n = 9 per group) of fibroblast invasion after HER2 knockdown. (G) Protein levels of p-HER2, total HER2, SEMA7A, and FOXF1 in IPF lung fibroblasts after treatment of HER2 inhibitor, Lapatinib, at increasing concentrations. (H) Downregulation of cell-surface expression of SEMA7A, F3, and ITGA6 in Lapatinib-treated fibroblasts was determined by flow cytometry analysis. (I) Transcription levels of other representative genes in IPF lung fibroblasts after Lapatinib treatment were determined by qRT-PCR ( n = 3 per group). Scale bar: 1 mm (E). CTL, control; KD, knockdown. Three or four independent experiments were performed on fibroblasts from different patients (A, C, F, and I). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student’s t test (A, C, and F) or one-way ANOVA (I). Source data are available for this figure: .

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, Control

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Blocking HER2 signaling inhibited IPF lung fibroblast invasion and attenuated fibrosis. (A–C) Representative images (A) and index quantification (B and C) of migration and invasion of fibroblasts treated with increasing doses of Lapatinib or DMSO ( n = 3 per group). (D–F) Representative images (D) and index quantification (E and F) of migration and invasion of fibroblasts treated with Pertuzumab or IgG1 ( n = 3 per group). (G and H) Masson’s trichrome staining of collagen in lung sections (G) and hydroxyproline content in lung tissues (H) from NSG mice injected with SEMA7A high IPF fibroblasts and treated with Lapatinib, vehicle control, Pertuzumab, or IgG1 control ( n = 10 per group). Dash-boxed regions were shown at higher magnification. Three independent experiments were performed on fibroblasts from different patients (B, C, E, and F). Data are the mean ± SEM. Scale bar: 1 mm (A and D) and 500 μm (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 by one-way ANOVA (B, C, E, and F) and two-way ANOVA (H).

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Blocking Assay, Migration, Staining, Injection, Control

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Targeting HER2 blunted bleomycin-induced murine lung fibrosis. (A and B) Fibroblast invasion (A) and invasion index (B) after combined treatment of Lapatinib and α-PDL1 ( n = 3 per group). (C and D) Masson’s trichrome staining (C) and hydroxyproline content of lung tissues (D) from C57BL/6J mice injured with 1.25 U bleomycin and treated with Lapatinib or vehicle control. Dash-boxed regions were shown at higher magnification (control [CTL] with vehicle, n = 9; CTL with Lapatinib, n = 8; bleomycin with vehicle, n = 9; bleomycin with Lapatinib, n = 7). Three independent experiments were performed on fibroblasts from different patients (B and D). Data are the mean ± SEM. Scale bar: 1 mm (A) and 500 μm (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 by two-way ANOVA (B and D).

Article Snippet: Immunofluorescence was performed using following primary antibodies overnight at 4°C: p-HER2 (6942s, clone D66B7; Cell Signaling Technology) and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology), and Cy3-conjugated secondary antibody and FITC-conjugated α-SMA antibody (ab8211, clone 1A4; Abcam) were used to visualize the staining.

Techniques: Staining, Control