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: ( A ) MAL3-101 sensitive (in blue) and resistant (in black) cells lines were transfected with a control siRNA or two different siRNAs that were directed against PERK or PKR. 72 hr post transfection cells were treated with DMSO or 12 μM MAL3-101 for 6 hr and lysates were prepared for immunoblot analysis. β-actin serves as a loading control, and the asterisk denotes a non-specific band in the Hsp70 blot. ( B ) PERK and PKR knockdown efficiency were measured by immunoblot 72 hr post siRNA transfection in MAL3-101 sensitive (MDA MB 231, closed circles) and resistant (MDA MB 453, open circles) cell lines. The relative fold change of PERK and PKR protein levels in knockdown to control samples is plotted ± SEM. *** denotes p<0.0005 and **** denotes p<0.00001. ( C ) MAL3-101 sensitive and resistant cells were treated with 12 μM MAL3-101, DMSO for 6 hr or with 10 μg/μl tunicamycin for 3 hr. PERK mobility was analyzed by immunoblot. ( D ) MAL3-101 sensitive (in blue) and resistant (in black) cells lines pre-treated with GSK-2606414 (GSK, 2 μM) for 2 hr prior to addition of DMSO or 12 μM MAL3-101 for 6 hr. Lysates were prepared for immunoblot analysis. β-actin serves as a loading control. Figure 10—source data 1. PERK and PKR raw quantification.

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: Transfection, Western Blot

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 ) MAL3-101 sensitive (MDA MB 231, in blue) and resistant (MDA MB 453, in black) cells were transfected with a control siRNA or two different siRNAs directed against GCN2, and 72 hr post transfection cells were treated with DMSO or 12 μM MAL3-101 for 6 hr. Lysates were immunoblotted to detect the indicated proteins. A non-specific band in the Hsp70 immunoblot is indicated with an asterisk (*), and β-actin served as a loading control. ( B ) GCN2 knockdown efficiency was measured by immunoblot 72 hr post siRNA transfection in MAL3-101-sensitive (MDA MB 231, closed circles) and MAL3-101-resistant (MDA MB 453, open circles) lines. The relative GCN2 fold change in knockdown to control samples is plotted, ± SEM (n≥3); **** denotes p<0.00001. ( C ) The corresponding fold increase of the indicated apoptotic markers and p-eIF2α in MAL3-101-sensitive (MDA MB 231, closed symbols) and MAL3-101-resistant (MDA MB 453, open symbols) cells treated with a control or siRNAs directed against GCN2 and treated with DMSO or MAL3-101 is shown, ± SEM (n≥3) * denotes p<0.05, *** denotes p<0.0005 and **** denotes p<0.00001. Figure 11—source data 1. Source data for .

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: Transfection, Western Blot

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: (1) In MAL3-101 resistant and sensitive cells, Hsp70 inhibition lead to activation of the ISR due to the accumulation of misfolded proteins (only misfolded proteins in the cytoplasm are shown). (2) GCN2-mediated eIF2α phosphorylation, which can be induced by amino acid starvation (see Discussion), also induces (3) the transcription of autophagy-related genes, favoring autophagy induction and (4) protecting MAL3-101-resistant breast cancer cells. (5) If GCN2 activity is impaired or (6) autophagy is inhibited (for example by chloroquine), Hsp70 inhibition is cytotoxic so (7) CHOP accumulates and resistant cells undergo apoptosis. (7) In contrast, in MAL3-101-sensitive cells, PERK induces ATF4 and CHOP, which results in cell death.

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: Inhibition, Activation Assay, Activity Assay

Journal: Scientific reports

Article Title: Megakaryocytes form linear podosomes devoid of digestive properties to remodel medullar matrix.

doi: 10.1038/s41598-022-10215-x

Figure Lengend Snippet: Figure 4. MKs lose their digestive ability together with Tks5 expression during maturation. (a) Lin− progenitors (P) and D3MKs were cultured for 6 h on gelatin- Oregon Green 488 (green) containing 100 µg/ml Horm collagen (non labelled) matrix prior to fixation and staining for F-actin (in red). (b) P and D3MKs were treated as in (a), then stained for F-actin (red) and Tks5 (green). (c) Quantification of mean Tks5 intensity per podosome from 3800 podosomes from 18 cells. Values are mean ± s.e.m.***P < 0.001 according to unpaired t-test. (d) P and D3MKs were treated as above, then stained for F-actin (red) and Tks5 linker domain (2G6 antibody) or (e) Tks5 PX domain (F4 antibody). Linescans show the distribution of fluorescence along the white line. Scale bars = 10 µm. n = number of cells studied.

Article Snippet: DAPI (1050, Euromedex), Alexa Fluor 633- 594- 488-conjugated phalloidin (A22284, A12381, A12379, Invitrogen), anti-active β1 (9EG7 BD Biosciences), DDR1 (5583T Ozyme), Tks5 (sc376211 Santa Cruz), Tks4 (09-260, Sigma Aldrich), HSC70 (sc59560 Santa Cruz), Talin (8d4, Sigma Aldrich), Arp2/3 complex subunit 2 (07-227, Sigma Aldrich), Vinculin (hVIN-1, V9264 Sigma Aldrich), CD44 (217594, Millipore), Col1-3/4C (0217-050, Immunoglobe), MT1-MMP (MAB3328, Millipore), β1- blocking antibody (’NA/ LE Hamster anti-rat CD29’ BD Biosciences, 555002).

Techniques: Expressing, Cell Culture, Staining, Fluorescence

Journal: Current biology : CB

Article Title: Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

doi: 10.1016/j.cub.2021.10.015

Figure Lengend Snippet: Figure 3. Archetypal analysis of MSN subtypes (A) Representative genes showing significant discontinuity between a subtype pair. (B) Representative genes showing non-significant discontinuity between a subtype pair. (C) The p value distributions between each subtype pair. (D) Heatmap showing the cosine similarity between cells within and between the nine types of MSNs. (E) FISH labeling of CRYM (magenta) and CNR1 (green) reveals a continuous gradient on the dorsal-ventral axis. (F) CRYM (top) and CNR1 (bottom) expressions along the D1/D2-hybrid archetype axes. (G) The archetype distribution of subtype pairs (top) that were divided between the dorsal and VS and CNR1 (bottom) distributions in these archetype axes. (H) Heatmap showing the cosine similarity between cells within and between striosome and matrix in Cd and Pt. The color scale is the same as in (D). (I) Distribution of Cd and Pt in archetype axes. (J) OPRM1 expression in the Cd and Pt cells along the archetype axes. See also Figure S3, Tables S1 and S2, and Data S2.

Article Snippet: To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH labeling with the two probes, we performed the following immunostaining with ChAT antibodies (ProSci, Cat# 45-037, 1:1000) using similar approach except that TBS buffer instead of PT buffer was used.

Techniques: Labeling, Expressing

Journal: Current biology : CB

Article Title: Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

doi: 10.1016/j.cub.2021.10.015

Figure Lengend Snippet: Figure 4. MSN subtypes in the dorsal striatum (A) FISH labeling of DRD1 (green) and DRD2 (magenta). White box indicates the region shown in (B). The top right axis shows D, V, L, and M di- rections. (B) High-resolution image of region highlighted in (A). (C) Schematic diagrams of the three sections used for DRD1 and DRD2 quantification. The square boxes indicate the quantified regions of interest (ROIs). (D) Quantification of cell density of neurons ex- pressing DRD1 (green), DRD2 (magenta), or both (orange) in the Cd and Pt. Error bars are SD across ROIs. (E) One example MSN expressing both DRD1 and DRD2. (F) RXFP1 labels D1/D2-hybrid MSNs in the dorsal striatum. Arrowhead points to an example D1/D2- hybrid cell. (G) Quantification of DRD1 and DRD2 grain number in D1/D2-hybrid cells and normal D1- or D2-MSNs. Unpaired t test was used for statistical analysis, and p values were indicated on the plots. Error bars represent standard deviation (SD) across 6 cells per type. NS, non-specific. (H) FISH labeling of DRD1 and RXFP1. Left two pictures are original FISH images showing the dis- tribution of DRD1 and RXFP1. The right two pic- tures are CellProfiler-processed images showing DRD1 expressing (red dots) or DRD1 and RXFP1 (black dots, enlarged for display purposes) co-ex- pressing cells. Gray dots are nuclei. (I) Immunohistochemistry of KChIP1 showing robust striosome pattern. (J) FISH labeling of KCNIP1 (yellow) and STXBP6 (blue) distinguishes striosome and matrix, respec- tively. White square indicated the region shown in (K). (K) Top: detail of the white square in (J). Bottom: as above, for characteristic striosome and matrix markers, BACH2 and GDA. Cd, caudate; Pt, putamen; IC, internal capsule. See also Figure S4, Table S1, and Data S4.

Article Snippet: To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH labeling with the two probes, we performed the following immunostaining with ChAT antibodies (ProSci, Cat# 45-037, 1:1000) using similar approach except that TBS buffer instead of PT buffer was used.

Techniques: Labeling, Expressing, Standard Deviation, Immunohistochemistry

Journal: Current biology : CB

Article Title: Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

doi: 10.1016/j.cub.2021.10.015

Figure Lengend Snippet: Figure 5. MSN subtypes in the VS (A) Top: double labeling with DRD1 (left) and ARHGAP6 (middle) of the NAs shell/OT shows that ARHGAP6 is selectively enriched in the shell/OT. Bottom: CellProfiler-processed images for the above images. Lettered boxes indicate regions shown in Figure S5A and S5B. (B) Left: quantification of grain number of ARHGAP6 in shell/OT and core. Unpaired t test was used for statistical analysis. Error bars represent SD across thirty- two cells per each region. Right: quantification of grain number of GREB1L in shell/OT and core. Unpaired t test was used for statistical analysis. Error bars represent SD across twenty-nine cells per each region. (C) Violin plot showing TAC3 levels in D1Sh-TAC3 archetype, other D1Sh cells, and TAC3 interneurons. (D) TAC3 co-localizes with DRD1 in medial shell MSNs. (E) DRD2 FISH image showing the outline of the striatum. Dashed white line delineate the borders of striatum. (F) CellProfiler results showing theTAC3 distribution (red dots) in the section in (E). Gray dots are nuclei. (G) CellProfiler results showing the distribution of TAC3 and DRD1 co-expressing cells (dark green dots) in the section in (E). (H) CellProfiler results showing the distribution of TAC3 and DRD2 co-expressing cells (dark green dots) in the section in (E). (I) CellProfiler results showing the distribution of TAC3 and DRD1 co-expressing cells in a second animal (dark green dots). See also Figure S5, Tables S1 and S3, and Data S3 and S4.

Article Snippet: To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH labeling with the two probes, we performed the following immunostaining with ChAT antibodies (ProSci, Cat# 45-037, 1:1000) using similar approach except that TBS buffer instead of PT buffer was used.

Techniques: Labeling, Expressing

Journal: Current biology : CB

Article Title: Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

doi: 10.1016/j.cub.2021.10.015

Figure Lengend Snippet: Figure 6. Cell types in the interface islands (A) FISH stain of DRD1 (green) and DRD2 (magenta). Inset: white area indicates striatum, and the red box highlights the area shown in (A) and (B). (B) FISH stain of DRD1, RXFP1, and CPNE4 in immediately adjacent section from (A). (C) High-resolution image of the regions indicated with the letter ‘‘C’’ in (B). (D) High-resolution image of the region indicated with the letter ‘‘D’’ in (B). (E) Distribution of RXFP1 and CPNE4 clusters across six rostral-caudal (R-C) regions identified by multichannel FISH. The upper right axis shows D, V, L, M, R, and C directions. Dashed white box denotes RXFP1 clusters in Pt, the FISH image of which is shown in Figure S5H. Yellow arrowheads point to RXFP1 clusters in caudal extent of NAc in both illustration and images shown in (F). (F) Example RXFP1 clusters in caudal extent of NAc. AC, anterior commissure; GPe, external globus pallidus; VP, ventral pallidum. See also Figures S5–S7, Table S1, and Data S4.

Article Snippet: To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH labeling with the two probes, we performed the following immunostaining with ChAT antibodies (ProSci, Cat# 45-037, 1:1000) using similar approach except that TBS buffer instead of PT buffer was used.

Techniques: Staining

Journal: Current biology : CB

Article Title: Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

doi: 10.1016/j.cub.2021.10.015

Figure Lengend Snippet: Figure 7. m-opioid receptor expression is specifically enriched in D1-NUDAP cells (A) FISH stain of DRD1 and RXFP1 as well as OPRM1 in two close sections. White dashed line delineates the boundaries of a RXFP1 cluster. (B) Quantification of grain number of OPRM1 in RXFP1 clusters and close D1-MSNs. Unpaired t test was used for statistical analysis. Error bars represent SD across forty-one cells in each group. (C) MOR expression in an adjacent section of (A). (D) FISH stain of DRD1 and CPNE4 as well as OPRM1 in two close sections. White dashed line delineates the boundaries of a CPNE4 cluster. (E) Quantification of grain number of OPRM1 in CPNE4 clusters and close D1-MSNs. Unpaired t test was used for statistical analysis. Error bars represent SD across thirty-six cells in each group. (F) MOR expression in an adjacent section of (D). See also Figure S7, Table S1, and Data S4.

Article Snippet: To verify DRD2 and CPNE4 labels cholinergic neurons, after FISH labeling with the two probes, we performed the following immunostaining with ChAT antibodies (ProSci, Cat# 45-037, 1:1000) using similar approach except that TBS buffer instead of PT buffer was used.

Techniques: Expressing, Staining