Journal: Cell death & disease

Article Title: Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

doi: 10.1038/s41419-021-04154-0

Figure Lengend Snippet: Fig. 8 Effects of S63845 and imatinib administered as single drug or in combination on the colony forming capacity of primary human CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase or from the bone marrow of healthy donors. Relative colony forming capacity of CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase (left panel; n = 6 for all S63845 concentrations) or from the bone marrow (BM) of healthy donors (right panel; n = 4 for all S63845 concentrations) in the absence or presence of 1 μM imatinib (IM) and/or the indicated concentrations of S63845. Data represent mean with range derived from four (CD34+ normal BM cells) to six (CD34+ CML cells) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Patient no. 1: ⚫; patient no. 2: ◼; patient no. 5: ◆; patient no. 6: ○; patient no. 8: △; patient no. 9: ▽; healthy donor no. 1 ⚫; donor no. 2: ◼; donor no. 3: ▲; donor no. 4: ▼.

Article Snippet: Primary human normal bone marrow CD34+ hematopoietic stem/ progenitor cells were obtained from American Type Culture Collection (Cat. No.: PCS-800-012) or from Lonza (Cat. No.: 2M-101), and kept in culture for 48 h in StemSpan SFEM II Medium supplemented with StemSpan CD34+ Expansion Supplement and 175 nM UM171 (all from STEMCELL Technologies) before starting the colony forming assay.

Techniques: Derivative Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USCs inhibited the proliferation of PBMNCs in MLR. USC-TA+, USC-TA−, BMSCs, and SMCs were used as stimulator cells and human PBMNCs from two healthy donors were used as responder cells. One-way MLR with single donor PBMNCs (donor A or B) as responder cells and two-way MLR with aliquot half dose of two different donors’ PBMNCs (donor A and B) as responder cells were detected in the same multi-well plate. Cultures were incubated at 37 °C for 5 days in 5% CO2 and 100% humidity, the cells were checked with inverted microscope on the 5th day before further experiment. There were different cell densities of PBMNCs be noticed in different wells for 5 days mix-culture, Scale bar =50 µm (A). The proliferation of PBMNCs was assessed with the BrdU cell proliferation colorimetric ELISA. Newly synthesized BrdU-DNA was quantified using a scanning multi-well spectrophotometer (B). *, P<0.05, post-hoc paired-comparisons. MLR, mixed lymphocyte reaction; BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells; SMC + PBMNC A, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor A; SMC + PBMNC B, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor B; SMC + PBMNC A + B, two-way mixed lymphocyte reaction as human smooth muscle cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; BMSC + PBMNC A, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor A; BMSC + PBMNC B, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor B; BMSC + PBMNC A + B, two-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; USC + PBMNC A, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor A; USC + PBMNC B, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor B; USC + PBMNC A + B, two-way mixed lymphocyte reaction as human urine derived stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Incubation, Inverted Microscopy, Enzyme-linked Immunosorbent Assay, Synthesized, Spectrophotometry, Derivative Assay, Activity Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USC showed a characteristic cytokine release profile. The supernatants from USC-TA+, USC-TA−, and BMSCs cultured alone or co-cultured with PBMNCs (direct mixed culture with PBMNCs, or indirect mixed culture with PBMNCs in transwell insert for 48 h) (A) were assessed for their relative levels of cytokines and chemokines by using the human cytokine array panel A, as described in “Methods” (B). A: human cytokine array panel template; B: PBMNCs background; C: USC-TA+ culture alone; D: USC-TA− culture alone; E: BMSCs culture alone; F: USC-TA+ direct mix culture with PBMNCs; G: USC-TA− direct mix culture with PBMNCs; H: BMSCs direct mix culture with PBMNCs; I: USC-TA+ in-direct mix culture with PBMNCs in transwell insert; J: USC-TA− in-direct mix culture with PBMNCs in transwell insert; K: BMSCs in-direct mix culture with PBMNCs in transwell insert: The immunoblot of cytokine expression levels were visualized and quantifed (C). *, P<0.05, comparing with BMSCs alone; †, P<0.05, comparing direct and indirect mixed culture with PBMNCs. BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Cell Culture, Western Blot, Expressing, Derivative Assay, Activity Assay

Journal: Development (Cambridge, England)

Article Title: Acid sphingomyelinase is a gatekeeper of placental labyrinthine architecture and function

doi: 10.1242/dev.204425

Figure Lengend Snippet: Increased autophagy in Smpd1 −/− placentas. (A) Densiometric analysis (left) of phospho-TFEB (p-TFEB) and total TFEB protein expression in gestational day (d) 15.5 wild-type and Smpd1 −/− placentas, normalized to β-actin. P-TFEB protein levels are significantly decreased in Smpd1 −/− placentas ( P <0.05). Although slightly elevated in wild-type placenta, there was no significant change in total TFEB levels. (Right) A TFEB immunoblot of wild-type (+/+) and Smpd1- deficient (−/−) d13.5, d15.5 and d17.5 placentas. (B) (Top) Immunofluorescence analysis of TFEB localization examined by confocal microscopy in d15.5 wild-type and Smpd1 −/− placentas at 40× magnification. TFEB (red) localizes to the cytoplasm of wild-type placental labyrinth and to the nucleus of labyrinth trophoblast in the Smpd1 −/− placental labyrinth. Lysosomes immunostained by LAMP1 (green) and nuclei are stained with DAPI (blue). Scale bars: 5 μm. (Bottom) Quantitation of TFEB localization in sinusoidal and syncytial cells. Smpd1 −/− placentas have a higher percentage of labyrinth trophoblasts, which have nuclear TFEB staining, compared to wild type ( P <0.05). (C) A phospho-mTOR (Ser2448) and a mTOR immunoblot (right) of wild-type and Smpd1 −/− d15.5 placentas, and densiometric analysis (left) of p-mTOR and mTOR protein expression normalized to β-actin. pmTOR protein level is significantly increased in Smpd1 −/− placentas. (D) A Phospho-p70S6K (Thr389) and p70S6K immunoblot (right) and densiometric analysis (left). All data are mean±s.e.m. per placenta obtained from at least two different dams. Different letters represent statistical significance ( P <0.05). For details of the statistical test(s) used, see the Materials and Methods.

Article Snippet: Primary antibodies used for western blotting were anti-β-actin, goat polyclonal (I-19) (Santa Cruz Biotechnology,sc-1616; 1:500), anti-LAMP-1 (DSHB, 1D4B; 1:1000), anti-TFEB (Bethyl, A303-673A; 1:2000), anti-mTOR Ser2448 (Cell Signaling, 2971; 1:800), anti-mTOR (Cell Signaling, 2972; 1:1000), anti-p62 (PROGEN Biotechnik, GP62-C; 1:1000), anti-LC3 (MBL, PM036; 1:1000), anti-ATP6V1B2 (Proteintech, 15097-1-AP; 1:1000), anti-Cathepsin D (Bio-Vision, 3191R-100; 1:2000), anti-DDDK-HRP (Proteintech, HRP-66008; 1:15,000), anti-p70 S6 kinase Thr389 (Cell Signaling, 9205; 1:800) and anti-p70 S6 kinase (Cell Signaling, 9202; 1:1000).

Techniques: Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Quantitation Assay

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were analyzed by Western blotting with antibodies as indicated. The levels of DRP1, FIS1, MFF, short form of OPA1(S-OPA1), long form of OPA1(L-OPA1), MFN1 and MFN2 in PINCH-1 KO A549 cells were quantified by densitometry and compared to those in A549 cells (normalized to 1) (right, n = 3). b PINCH-1 KO A549 cells were infected with lentiviral vectors encoding 3f-P1 or 3 f. Three days later, cells (as indicated) were analyzed by Western blotting. DRP1 level in indicated samples was quantified by densitometry and compared to A549 (right, n = 3). c H1299 cells were infected with Sh-P1 or Sh-con lentivirus and analyzed by Western blotting with antibodies as indicated. The levels of DRP1 in PINCH-1 knockdown H1299 cells were quantified by densitometry and compared to those in H1299 cells (normalized to 1) (right, n = 5). d DRP1 mRNA levels in A549 cells (as indicated) were analyzed by RT-PCR ( n = 3). e DRP1 mRNA levels in H1299 cells (as indicated) were analyzed by RT-PCR ( n = 6). f A549 cells were infected with ILK shRNA (Sh-ILK) or Sh-con lentivirus and analyzed by Western blotting as indicated. The levels of DRP1 in ILK knockdown cells were quantified by densitometry and compared to those in A549 cells (normalized to 1) (right, n = 4). g Mitochondria were stained with MitoTracker Red CMXRos and the percentages of mitochondria with different morphologies were quantified (right, n = 30 cells). Bar, 5 μm. h Mitochondrial areas in z-stack images were quantified ( n = 31 cells). i Wild type and kindlin2 (K2) KO A549 cells were analyzed by Western blotting with antibodies recognizing DRP1, P1 or tubulin. The level of DRP1 in kindlin2 KO A549 cells was quantified by densitometry and compared to that in wild type A549 cells (normalized to 1) (right, n = 4). Data represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. Source data are provided as a Source Data file. The samples in a , b , c , f , and i were from the same experiment and the blots were processed in parallel.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Western Blot, Infection, Knockdown, Reverse Transcription Polymerase Chain Reaction, shRNA, Staining

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were transfected with siRNA targeting DRP1 (Si-DRP1) or control siRNA (Si-con) as indicated. Three days later, the cells were analyzed by Western blotting with antibodies recognizing DRP1, P1, or tubulin. The samples were from the same experiment and the blots were processed in parallel. Mitochondria were stained with MitoTracker Red CMXRos ( b ) and the percentages of mitochondria with different morphologies were quantified ( c ) (A549 n = 43 cells, P1KO n = 70 cells, P1KO + Si-Con n = 77 cells, P1KO + Si-DRP1 n = 55cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Bar, 5μm. d Mitochondrial areas in z-stack images were quantified (A549 and P1KO and P1KO + Si-con n = 32 cells, P1KO + Si-DRP1 n = 30 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). e , f Mitochondria (red arrows) were observed under TEM ( e , bar, 500 nm) and the areas of mitochondria were quantified ( f , n = 40 mitochondria; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P = 0.0028). The cells were stained with DAPI (blue) and antibody for Ki67 (purple) ( g ) and the percentages of Ki67-positive cells were quantified ( h , A549 and P1KO n = 33 fields, P1KO + Si-con and P1KO + Si-DRP1 n = 31 fields; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Bar, 25 μm. i The cells were cultured for 4 days and the numbers of live cells were analyzed as in Fig. ( n = 4; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Data in c , d , f , h , and i represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Transfection, Control, Western Blot, Staining, Cell Culture

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were transfected with Si-DRP1 or Si-con as indicated. Three days later, cells (as indicated) were analyzed by Western blotting with antibodies recognizing PYCR1, DRP1, PINCH-1, or tubulin. PYCR1 level was quantified by densitometry (right, n = 4; A549 vs P1KO P = 0.0076, A549 vs P1KO + Si-con P = 0.0023, P1KO vs P1KO + Si-DRP1 P = 0.0419, P1KO + Si-con vs P1KO + Si-DRP1 P = 0.012). b The cytosolic (Cyto) (lane 5, 7, 9 and 11), mitochondrial (Mito) (lane 6, 8, 10, and 12) and total (lane 1, 2, 3, and 4) fractions from the cells were analyzed by Western blotting with antibodies recognizing kindlin-2 (K2), PHB2, or tubulin. Kindlin-2 level in mitochondria was quantified (right, n = 3; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). c Cells were analyzed by PLA with kindlin-2 and PYCR1 antibodies. Bar, 10 μm. The number of PLA dots per cell were counted (right, n = 40 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). d Cells (as indicated) were analyzed by IP and Western blotting. Lane 2, the sample was prepared as that of lane 3 except anti-kindlin-2 antibody was substituted with irrelevant mouse IgG. PYCR1 level was quantified by densitometry (right, n = 4; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). e The proline level was analyzed using the absorbance method as described in the “Methods” (right, n = 3 independent experiments; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P = 0.0086, P1KO + Si-con vs P1KO + Si-DRP1 P = 0.0058). A representative set of samples were shown in the left. Bar, 1 cm. f A549 cells were infected with lentiviral vector encoding 3xFLAG-tagged MFN2 (3f-MFN2) or with 3xFLAG vector (3f). Three days later, the cells were analyzed by PLA with kindlin-2 and PYCR1 antibodies. Bar, 10 μm. The number of PLA dots per cell were counted (right, A549 n = 34 cells, 3f n = 33 cells, 3f-MFN2 n = 46 cells; A549 vs 3f-MFN2 P = 0.0074, 3f vs 3f-MFN2 P = 0.0027). g The cytosolic (lane 4, 6, and 8), mitochondrial (lane 5, 7, and 9) and total (lane 1, 2, and 3) fractions from the cells were analyzed by Western blotting. The levels of kindlin-2 in the cytosolic or mitochondrial fractions were quantified by densitometry. Right panel, the ratio of the mitochondrial kindlin-2 level divided by the cytosolic kindlin-2 level in the MFN2 overexpressing cells was compared to that in the control infectants or wild type A549 cells (normalized to 1, n = 3; A549 vs 3f-MFN2 P < 0.0001, 3f vs 3f-MFN2 P < 0.0001). Data in a – g represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; ** P < 0.01; *** P < 0.001. The samples in a , b , d , and g were from the same experiment and the blots were processed in parallel. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Transfection, Western Blot, Infection, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were infected with 3xFLAG-PYCR1 (3f-PY1) or 3xFLAG (3f) lentivirus. The cells were analyzed by Western blotting with antibodies as indicated. DRP1 level was quantified (right, n = 3; A549 vs P1KO P = 0.006, A549 vs P1KO + 3f P = 0.013, P1KO vs P1KO + 3f-PY1 P = 0.019, P1KO + 3f vs P1KO + 3f-P1 P = 0.0434). The samples were from same experiment and blots were processed in parallel. b The proline level was analyzed using the absorbance method (right, n = 3 independent experiments; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). A representative set of samples were shown in the left. Bar, 1 cm. c The numbers of live cells (A549 cells, red line; P1 KO cells, orange line; 3f lentiviral vector infected P1 KO cells, blue line; 3f-PY1 lentiviral vector infected P1 KO cells, green line) were analyzed as in Fig. ( n = 3). d Cells were stained with DAPI (blue) and Ki67 antibody (purple). Bar, 25 μm. The percentages of Ki67 positive cells were quantified (right, A549 n = 56 fields, P1KO n = 33 fields, P1KO + 3f n = 42 fields, P1KO + 3f-PY1 n = 51 fields; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). e Mitochondria were stained with MitoTracker Red CMXRos and mitochondria with different morphologies were quantified (right, A549 and P1KO n = 50 cells, P1KO + 3f n = 40 cells, P1KO + 3f-PY1 n = 37 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). Bar, 5 μm. f Mitochondrial areas in z-stack images were quantified (A549 n = 33 cells, P1KO n = 35 cells, P1KO + 3f n = 36 cells, P1KO + 3f-PY1 n = 37 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). g Mitochondria (red arrows) were observed under TEM (left panels, bar, 500 nm) and mitochondria areas were quantified (right panel, n = 40 mitochondria; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P = 0.001). Data in a – g represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; *** P < 0.001. Source data are provided in Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Infection, Western Blot, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: The lung of the mice was administrated with Ad-Cre and analyzed 16 weeks later. a Sections of the lung tissues from the mice (as specified in the figure) were analyzed by immunostaining with antibodies for PINCH-1 (P1)(top), DRP1 (middle), or PYCR1 (bottom). Bar, 20 μm. The boxed areas in the IHC staining were enlarged and shown in the upper right corner. Right panels, the mean intensities of PINCH-1, DRP1, and PYCR1 staining in the Kras LSL−G12D/+ ; PINCH-1(P1) fl/fl (Kras fl/+ ; P1 fl/fl ) group were quantified and compared to those of the Kras fl/+ group (normalized to 1; n ≥ 30 fields from 6 mice for each group; different mice in each group were coded with different colors; P < 0.0001). b The DRP1 mRNA levels from the lung tissues (as indicated) were analyzed by RT-PCR ( n = 4; P = 0.0307). c The proline levels in the lung tissues were analyzed as described in the Methods ( n = 8 mice; P < 0.0001). d Collagen matrix was analyzed by SHG with multiphoton microscopy (top; bar, 100μm), immunostaining with antibody for collagen1A1 (middle; bar, 20 μm), or Masson’s trichrome staining (bottom; bar, 20 μm). The boxed areas in the IHC staining were enlarged and shown in the upper right corner. Bottom panels, for each method (as indicated in the figure) the mean intensities of collagen matrix in the Kras fl/+ ; P1 fl/fl group were quantified and compared to those of the Kras fl/+ group (normalized to 1; n = 30 fields from 6 mice for each group; different mice in each group were coded with different colors; P < 0.0001). Data in a – d represent mean ± SEM. Statistical significance was calculated using two-tailed unpaired Student’s t -test, * P < 0.05; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Immunostaining, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction, Microscopy, Two Tailed Test